Detecting microbial infection in wounds

ABSTRACT

The disclosed technology relates to chemical entities for the detection of wounds, e.g., chronic wounds or infected wounds, including compositions, substrates, kits, dressing materials, and articles, and systems containing such compounds. The disclosed technology further relates to methods of using these compositions, kits and systems in diagnostic assays, and in the diagnosis and/or detection of chronic or infected wounds based on enzymatic action on specific moieties and/or reaction sites. The disclosed technology additionally relates to detection of pathogenic, e.g., bacterial and/or viral substances, such as enzymes and substrates, at the wound situs. Additional disclosure relates to methods of characterizing wounds based on expression of a plurality of markers and using such information to treat, manage, and follow-up patients suffering from chronic or infected wounds.

CROSS-REFERENCE TO RELATED APPLICATIONS AND DISCLOSURE

This application claims the benefit of United States ProvisionalApplication Nos. 62/315,546, filed Mar. 30, 2016, and U.S. 62/315,556,filed Mar. 30, 2016, which disclosures are incorporated herein byreference in their entireties and made a part hereof.

SEQUENCE LISTING

The Sequence Listing associated with this application, which is separatepart of the disclosure, includes the nucleotide and/or amino acidsequences and associated information using the symbols and format inaccordance with the requirements of 37 CFR 1.821-1.825. The SequenceListing in the XML file titled CVT05-42222 ST.26 Version.xml, which wascreated on Sep. 19, 2023 and is 47 kilobytes, is incorporated herein byreference.

TECHNICAL FIELD

Embodiments described herein generally relate to wound healing, and inparticular to compositions and methods for the detection and treatmentof wounds.

BACKGROUND

In mammals, dermal injury triggers an organized complex cascade ofcellular and biochemical events that result in a healed wound. Woundhealing is a complex dynamic process that results in the restoration ofanatomic continuity and function: an ideally healed wound is one thathas returned to normal anatomic structure, function, and appearance. Atypical wound heals via a model consisting of four stages—“exudative”phase, proliferative phase, reparative phase and epithelial maturation(Hatz et al., Wound Healing and Wound Management, Springer-Verlag,Munich, 1994) or hemostatic, inflammatory, proliferative and remodelingphase (Nwomeh et al., Clin. Plast. Surg. 1998, 25, 341). Theinflammatory phase is particularly important to the wound healingprocess, wherein biochemical reactions at the wound situs facilitatehealing but also cause tissue breakdown due to production of excessproteases.

Pathogenic infection is one of the most common impediments to woundhealing. A progressive worsening of a clean wound to a colonized woundis often associated with increased bioburden imposed by pathogenicmicroorganisms. See, Ovington et al., Ostomy Wound Management,49.7A:8-12, 2003. An infected wound is an intermediate stage that ischaracterized by clinical signs of infection such as yellow appearance,soreness, redness, oozing pus, while a colonized wound is characterizedby chronic pathogenic infection and is difficult to heal. Infection ofthe wound may also arrest the healing process. For example, pathogens ina wound can produce toxins (e.g., Clostridium species), generate noxiousmetabolites like ammonia that raise pH (e.g., Proteus species), activateor produce tissue lytic enzymes like proteases, or promote tissueinvasion, thereby leading to an increase in the size or seriousness ofthe wound.

In order to keep the chronicity of wounds in check, a variety ofassessment techniques and/or tools are employed in the clinical andveterinary setting. Current methods of assessing an infected wound arebased primarily on assaying for a variety of parameters associated withthe wound. For instance, a wound may be assessed visually, length anddepth measurements may be taken, digital photography may be used whereavailable to track the visual condition and size of a wound (Krasner etal., supra). In clinical practice, diagnosis of infection is based onmeasurement of secondary parameters, such as, odor, presence of localpain, heat, swelling, discharge, and redness. Many of these clinicalindicators, such as inflammation and discharge have a low predictivevalue of infection in wounds. In other instances, the number(s) andtype(s) of pathogenic flora at the wound situs may be determined usinglaboratory and/or clinical diagnostic procedures. Swabbing of a woundfollowed by microbiology testing in the hospital laboratory is an optionfor confirmation of bacterial colonization and identification of thestrains associated with infection, thus allowing for the prescription ofcorrect antibiotic course. However, this process is time consuming andlabor intensive. Delay in diagnosis of infection can delay theadministration of antibiotics and may increase the risk of developingsepsis.

One of the biggest drawbacks associated with existing clinicaldiagnostics is a lag associated with the onset of infection and thetiming of detection. For instance, positive identification of infectionusing swabbing procedures often depends on attainment of a “criticalmass” of microorganisms at the wound site and so early detection cannotbe made until a detectable level is reached. Also, the swabs may becontaminated with the flora of the surrounding tissue, therebycomplicating the diagnostic procedure. Other drawbacks include, e.g.,sampling errors, delays in transport of the swabs, errors in analyticalprocedures, and/or errors in reporting. See, the review by Bowler etal., Clin Microbiol Rev. 14(2): 244-269, 2001.

There is therefore an imminent but unmet need for diagnostic reagentsand methods that enable early diagnosis of clinical infection,preferably, which permit clinical diagnosis prior to manifestation ofclinical symptoms of infection. There is also a need for compositionsand methods that would assist in predicting clinical infection of awound prior to the manifestation of clinical symptoms. Such a prognosticaid would allow early intervention with suitable treatment (e.g.,antimicrobial treatment) before the wound is exacerbated and surgery orother drastic intervention is required to prevent further infection.Additionally, if clinicians could respond to wound infection as early aspossible, the infection could also be treated with minimal antibioticusage. This would reduce the need for hospitalization and would reducethe risk of secondary infections, e.g., as a result of contact withother diseased subjects.

SUMMARY

The technology disclosed herein provides for compositions and methods ofdetecting infected and/or chronic wounds. The disclosed technologyimproves upon exiting assays by: increasing the sensitivity, precisionand specificity of detection of infected wounds; providing for theability of qualitative and quantitative measurements; and, increasingthe speed of detection of infected wounds in situ and in real-time. Theassays and methods described herein are partly based on the use ofspecific reagents that detect biomarkers and/or probes which are presentin infected or chronic wounds. The detection process may involve use ofreagents that are specific to the markers present in infected wounds butnot non-infected or non-chronic wounds and the detection step mayinvolve qualitative or quantitative measurements of the signal(s) thatare generated when the probe is acted upon by the marker. In embodimentswherein the detection method involves detection of enzymes present inwounds, the probes comprise modified enzyme substrates that are specificto the enzyme, which generate signals that may be optionally amplified.This greatly improves efficiency and specificity of detection. Moreover,a plurality of detection probes, each specific for one or more targets,e.g., enzymes that are specific to the wounds, may be employed. Thisgreatly helps to maximize both efficiency and accuracy of diagnosticassays while minimizing the incidence of false positives {e.g., duenon-specific interactions and/or target redundancy). Furthermore, theexperimental results disclosed herein confirm that the novel probes andthe assay techniques based thereon are capable of detecting andcharacterizing various types of wounds. Finally, the reagents of thedisclosed technology may be used together with therapeutic moleculessuch as antibiotics, antifungal agents, etc. to monitor and evaluatetreatment and management of chronic wounds.

Embodiments described herein are based, in part, on the discovery thatcells of the immune system, including enzymes generated thereby, mayserve as markers in the early diagnosis of wounds. These cells, e.g.,neutrophils, are recruited at the wound situs to combat infection, do soby engulfing bacteria (and other pathogens) and/or neutralizing themwith enzymes. Some enzymes are specific towards proteins (e.g.,elastase, cathepsin G, lipase), others are specific towards cell wallcomponents (e.g., lysozyme) and yet others mediate protein denaturation(e.g., NADPH oxidase, xanthine oxidase, myeloperoxidase (MPO) and otherperoxidases). These cells, e.g., neutrophils, are generally onlyshort-lived and when they lyse in the area of the infection, theyrelease the contents of their lysosomes including the enzymes, which canthen be detected to provide a reliable measurement of the status of thewound.

Accordingly, various embodiments described herein utilize the detectionof enzyme markers, which are indicative of the presence of myeloidcells, and neutrophils in particular, in a biological sample ofinterest, for example, wound tissue. Increased level or activity of suchenzymes in the wound fluid, therefore, corresponds to a heightenedbacterial challenge and a manifestation of disturbed host/bacteriaequilibrium in favor of the invasive bacteria.

In one aspect, provided herein is a chemical entity capable of detectingenzyme activity from a body fluid, the chemical entity comprising one ormore of an anchor region, an enzyme recognition region, an enzyme-labileor enzyme-reactive region, and an indicator region.

In some embodiments, the chemical entity comprises at least three of ananchor region, an enzyme recognition region, an enzyme-labile orenzyme-reactive region and an indicator region. In some embodiments, thechemical entity comprises one of an anchor region, an enzyme-labile orenzyme-reactive region and an indicator region. In some embodiments, thechemical entity comprises one of an anchor region, one of an enzymerecognition region, one of an enzyme-labile or enzyme-reactive regionand one of an indicator region. In some embodiments, the chemical entitybinds to a support material via the anchor region. In some embodiments,the chemical entity comprises at least two indicator regions. In someembodiments, the enzyme recognition region partially or fully overlapswith the enzyme-labile or enzyme-reactive region. In some embodiments,the anchor region partially or fully overlaps with the enzyme-labile orenzyme-reactive region. In some embodiments, the anchor region partiallyor fully overlaps with the indicator region. In some embodiments, theindicator region partially or fully overlaps with the enzyme-labile orenzyme-reactive region. In some embodiments, the indicator region, onceseparated from the chemical entity by target enzyme activity, interactswith one or more accessory enzymes selected from a lipase, esterase,peroxidase, oxidase, glycosidase, glucuronidase, glucosidase,galactosidase, and a combination thereof. In some embodiments, theenzyme-labile or enzyme-reactive region interacts with one or moretarget enzymes selected from Napsin (aspartyl protease),Glucosylceramidase glucuronidase, palmitoyl protein thioesterase,Cathepsins A, B, D, G, L, S, Z, Acid ceramidase, lactoferrin (LF),lysozyme, myeloperoxidase (MPO), elastase, cathepsins, and proteinase-3elastase, lysozyme, esterase, lipase and, and a combination thereof.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a moiety capable of producing a visible color or detectableelectronic change upon interaction of the enzyme-labile orenzyme-reactive region with one or more target enzymes, the moiety beingselected from a peroxidase substrate, arylamine, an amino phenol, aphenol, a quinone, a neutral dye, a charged dye, a nanoparticle, aquantum dot, a colloidal gold particle, or an analog thereof. In someembodiments, the peroxidase substrate is selected from p-aminophenol,ABTS (2,2inophenol, ABTS (rate is selected from gold acid) diammoniumsalt), 3,3′-diaminobenzidine, DCPIP, N,N-dimethyl-p-phenylenediamine,o-dianisidine, p-phenylenediamine, 4-chloro-1-naphthol,o-phenylenediamine N-(4-aminobutyl)-N-ethylisoluminol,3-amino-9-ethylcarbazole, 4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), indoxyl, indigo,Fast Blue RR, and 4-chloro-7-nitrobenzofurazan, and an analog thereof.In some embodiments, the peroxidase substrate an aniline analog. In someembodiments, the peroxidase substrate is an N-alkyl derivative of FastBlue RR with more than 6 carbon units. In some embodiments, theindicator region comprises a moiety capable of producing a visible coloror detectable electronic change upon interaction of the enzyme-labile orenzyme-reactive region with one or more target enzymes, the moiety beingselected from an indoxyl analog, a neutral dye, a charged dye, ananoparticle, and a colloidal gold particle. In some embodiments, themoiety capable of producing a visible color or detectable electronicchange is a charged dye or a luminol derivative. In some embodiments,the charged dye is selected from toluidine blue, reactive black 5,remazol brilliant blue, reactive violet 5, and reactive orange 16. Insome embodiments, the charged dye is selected from reactive blue 4,reactive red 120, reactive blue 2, reactive green 19, and reactive brown10. In some embodiments, the enzyme-labile or enzyme-reactive region islabile to or reactive with lysozyme, and the enzyme-labile or enzymereactive region comprises a polysaccharide, glucosamine, orpeptidoglycan, and the polysaccharide, glucosamine, or peptidoglycan. Insome embodiments, the enzyme-labile or enzyme-reactive region comprisesa peptidoglycan, and peptidoglycan is labile to or reactive withlysozyme. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises a phenol, a napthol, an indoxyl, or a quinone, and thephenol, carboxyaminophenyl, indoxyl, or quinone is labile to or reactivewith myeloperoxidase and not reactive to heme. In some embodiments, theenzyme-labile or enzyme-reactive region comprises a peptide,peptidomimetic, or protein, and the peptide, peptidomimetic, or proteinis labile to or reactive with elastase. In some embodiments, theenzyme-labile or enzyme-reactive region comprises a peptide comprisingan amino acid sequence of X_(y)AAPX_(y)-L-Z, wherein each X isindependently any amino acid, each y is independently a number selectedfrom 1 to 50, L is a linking moiety, and Z is a moiety capable ofcausing a visible color change or a detectable electronic change. Insome embodiments, the enzyme-labile or enzyme-reactive region comprisesa peptide comprising an amino acid sequence of X_(y)AAPVX_(y)-L-Z [SEQID NO: 1], wherein each X is independently any amino acid, each y isindependently a number selected from 0 to 50, L is a linking moiety suchas an ester or amide, and Z is a moiety capable of causing a visiblecolor change or a detectable electronic change. In some embodiments, theenzyme-labile or enzyme-reactive region comprises a peptide,peptidomimetic, or protein, and the peptide, peptidomimetic, or proteinis labile to or reactive with cathepsin G. In some embodiments, theenzyme-labile or enzyme-reactive region comprises a peptide comprisingan amino acid sequence of X_(y)N⁴N³N²N¹X_(y)-L-Z, wherein each X isindependently any amino acid; each y is independently a number selectedfrom 0 to 6; N⁴ is selected from alanine, glycine, valine, andglutamine; N³ is selected from alanine, glycine, proline, lysine, andserine; N² is selected from proline, alanine, and glycine; N¹ isselected from serine, lysine, phenylalanine, arginine, leucine, andmethionine; L is a linking moiety, and Z is a moiety capable of causinga visible color change or a detectable electronic change. In someembodiments, the anchor region is selected from a polystyrene bead,silica gel bead, polysaccharide bead, polyacrylamide bead, cellulosebead, polysaccharide, derivatized cellulose, polyacrylate,polyethyleneimine, polyacrylamide, UV-activatable group, phenolic azide,epoxide, peptidoglycan, an aliphatic chain, an aliphatic alcohol chain,a multi-cyclic or multi-aromatic ring system, a lipophilic group, and acombination thereof. In some embodiments, the anchor region binds to asupport material after a short period of UV irradiation. In someembodiments, the anchor region comprises an ionic chemical group forbinding to a support material. In some embodiments, the anchor regioncomprises a reactive moiety for covalent attachment to the supportmaterial. In some embodiments, the anchor region and the enzyme labileregion are polypeptides and the anchor region comprises a polymerbinding domain. In some embodiments, the enzyme labile region is labileto a protease and the polymer binding domains are selected fromhydrophobic binding domains. In some embodiments, the enzyme labileregion is labile to cathepsin or elastase. In some embodiments, thechemical entity is selected from a small molecule entity or a modifiedpolymer.

In one aspect, provided herein is a chemical entity for the detection ofinfection, the chemical entity comprising an indicator region comprisinga pH-sensitive moiety that presents a visible color change. In someembodiments, the chemical entity further comprises a reactive group thatallows the reaction to a solid phase. In some embodiments, thepH-sensitive moiety is bromothymol blue, phenol red, bromophenol red,chlorophenol red, thymol blue, bromocresol green, bromocresol purple; orother sulfophthalein dyes.

In some embodiments, the anchor region binds to a support material aftera short period of UV irradiation. In some embodiments, the anchor regioncomprises an ionic chemical group for binding to a support material. Insome embodiments, the anchor region comprises a reactive moiety forcovalent attachment to the support material. In some embodiments, theanchor region comprises a hydrophobic moiety that causes little or nosolubility in aqueous systems thus allowing the material to stayassociated with a solid phase.

In some embodiments, the reactive region interacts with the bacterialenzyme β-lactamase. This is a bacterial enzyme that is capable ofdegrading common antibacterial drugs and its presence is of interest totreating physicians. A chromogenic β-lactamase substrate is generallyuseful in addition to reagents that report on other biomarkers.

In some embodiments, the reactive region is a substrate for viralproteases or the host furin protease. In some embodiments, the detectionof the cleaved product requires a polycationic trap. In certainembodiments, these can be cross-linked. Depending on the degree and typeof cross-linking they can also be superabsorbers. In some embodiments,the enzyme substrates are able to give rise to a redox active speciesthat can be detected electronically. In other embodiments, theelectronic detection of enzyme products in solid phase is made by meansof reflected light. Disclosed herein are chemical entities, which can beof monomelic, oligomeric or polymeric nature. These are modified toserve as medium for detection of chosen marker for infection in a woundor in body fluids, before the infections are otherwise apparent. In someembodiments, the chemical entity is for detecting infection in a mammal.In some embodiments, the chemical entity detects one or more biomarkersof infection and produces a visible change in the presence of the one ormore biomarkers. In some embodiments, the one or more biomarkers areleukocyte enzymes. In some embodiments, the one or more biomarkers areselected from elastase, lysozyme, myeloperoxidase, leukocyte peroxidase,esterase, lipase, napsin (aspartyl protease), glucosylceramidaseglucuronidase, palmitoyl protein thioesterase, cathepsins A, B, D, G, L,S, Z, Acid ceramidase, lactoferrin (LF), and proteinase-3, β-lactamase,and other similar enzymes, or combination thereof. In some embodiments,the chemical entity detects a specific pH range. In some embodiments,the chemical entity detects one or more leukocyte enzymes and a specificpH range; and produces a visible change in the presence of the one ormore leukocyte enzymes and the specific pH range. In some embodiments,the visible change is a color change that is easily distinguished fromcolors common in wounds or body fluids (e.g., red, yellow, pink, orbrown). In some embodiments, the visible change is fluorescent,luminescent, or mediated via physical means such as refraction, gasevolution, or a change in polymer state. In some embodiments, thechemical entity comprises one or more components selected from the groupconsisting of: an anchor region, an enzyme recognition region, anenzyme-labile or enzyme-reactive region, and an indicator region. Insome embodiments, the body fluid is blood, plasma, serum, cerebrospinalfluid, sputum, urine or wound exudate. In preferred embodiments, thebody fluid is wound exudate.

In some embodiments, the chemical entity is incorporated into a wounddressing where the chemical entity reacts with wound exudates that comeinto contact with the wound dressing. In some embodiments, the chemicalentity is incorporated into a wound dressing where the chemical entityreacts with wound exudates that are drawn up through the wound dressingto a reagent layer comprising the chemical entity. In some embodiments,the chemical entity is an indicator conjugate that gives rise to a coloror other visible marker when infection biomarkers are present. In someembodiments, the chemical entity is used in methods to diagnose aninfected wound in a mammal. In some embodiments, the chemical entity isused in methods to treat a wound in a mammal. In some embodiments, thechemical entity is used in methods to diagnose and treat a wound in amammal.

In some embodiments, the chemical entity is incorporated into a vacumewound therapy system via tubing or other components where the chemicalentity reacts with vacume exudates that come into contact with theinsert in the device.

In some embodiments, the chemical entity is incorporated into aventilator system via tubing or other components where the chemicalentity reacts with aspirates, aerosols or sputum exudates that come intocontact with the chemical entities.

In some embodiments, the chemical entity is incorporated into a dipstickwhere the chemical entity reacts after the fluids to be assess areapplied using an external swab or similar. In some embodiments, thechemical entity gives rise to a change that can be detectedelectronically either via reflected light, amperometry or a similarelectrochemical process.

In some embodiments, use of the chemical entity makes it feasible todetect changes in the infection status of a mammal or patient prior tothese changes being otherwise apparent. In some embodiments, the methodsare the basis for an improved or proactive therapy, wherein a subsequentchange of treatment or an application of a more detailed diagnostic issubsequently used to select a therapy to prevent a worsening of themedical condition of a mammal or patient.

Chemical Entity

In some embodiments, the chemical entity is a small molecule chemicalentity or a modified polymer comprising one or more components selectedfrom the group consisting of: an anchor region, an enzyme recognitionregion, an enzyme-labile or enzyme-reactive region, and an indicatorregion.

In some embodiments, the enzyme-labile or enzyme-reactive region is astructure that is reacted by an enzyme. In some embodiments, the enzymerecognition site is a structure that allows binding to an enzyme.

In certain embodiments, the chemical entity is a modified polymer. Incertain embodiments, the chemical entity is a small molecule chemicalentity. Disclosed herein, are chemical entities comprising one or morecomponents selected from the group consisting of: an anchor region, anenzyme-labile or enzyme-reactive region, an enzyme recognition region,and an indicator region. In some embodiments, the chemical entitycomprises at least one anchor region, at least one enzyme recognitionregion, at least one enzyme-labile or enzyme-reactive region, and atleast one indicator region. In some embodiments, the chemical entitybinds to a support material through the anchor region. In someembodiments, the chemical entity is an indicator conjugate. In someembodiments, the chemical entity comprises at least one enzyme-labile orenzyme-reactive region, at least one enzyme recognition region and atleast one indicator region. In some embodiments, the chemical entitycomprises at least one anchor region, and at least one indicator region.

In some embodiments, the chemical entity comprises an anchor region, anenzyme recognition region, an enzyme-labile or enzyme-reactive region,and an indicator region. In some embodiments, the chemical entitycomprises an enzyme recognition region, an enzyme-labile orenzyme-reactive region, and an indicator region. In some embodiments,the chemical entity comprises an anchor region, an enzyme recognitionregion, and an enzyme-labile or enzyme-reactive region. In someembodiments, the chemical entity comprises an enzyme recognition region,an anchor region and two enzyme-labile or enzyme-reactive regions. Insome embodiments, the chemical entity comprises an enzyme recognitionregion, an anchor region, two enzyme-labile or enzyme-reactive regions,and two indicator regions. In some embodiments, the chemical entitycomprises an enzyme recognition region, two enzyme-labile orenzyme-reactive regions, and two indicator regions.

In some embodiments, the one or more anchor regions and the one or moreenzyme-labile or enzyme-reactive regions overlap partially with oneanother. In some embodiments, the anchor region and the enzymerecognition region or the enzyme-labile or enzyme-reactive regionpartially or fully overlap with one another. In some embodiments, theenzyme recognition region and the enzyme-labile or enzyme-reactiveregion partially or fully overlap with one another. In some embodiments,the one or more anchor regions are within the one or more enzyme-labileor enzyme-reactive regions. In some embodiments, the anchor region iswithin the enzyme-labile or enzyme-reactive region. In some embodiments,the one or more enzyme-labile or enzyme-reactive regions are within theone or more anchor regions. In some embodiments, the enzyme-labile orenzyme-reactive region is within the anchor region.

In some embodiments, the anchor region of the chemical entity binds thechemical entity to a support material. In some embodiments, the anchorregion comprises an ionic chemical group. In some embodiments, the ionicchemical group forms an ionic bond with the support material. In someembodiments, the anchor region comprises a hydrophobic moiety. In someembodiments, the hydrophobic moiety interacts with the support materialto bind the chemical entity to the support material. In someembodiments, the anchor region comprises a hydrophilic moiety. In someembodiments, the hydrophilic moiety interacts with the support materialto bind the chemical entity to the support material.

In some embodiments, the anchor region is a bead, a polymer, a materialwith an ionic chemical group, a material with a hydrophilic moiety, or amaterial with a hydrophobic moiety. In some embodiments, the anchorregion is a bead. In some embodiments, the anchor region is a polymer.In some embodiments, the anchor region is a material with an ionicchemical group, wherein the ionic chemical group is positively charged.In some embodiments, the anchor region is a material with an ionicchemical group, wherein the ionic chemical group is negatively charged.In some embodiments, the anchor region is a material with a hydrophilicmoiety. In some embodiments, the anchor region is a material with ahydrophobic moiety such as an aliphatic chain or an aliphatic alcohol.In some embodiments, the anchor region comprises a reactive moiety forcovalent attachment to a support material such as a photoactivephenylazide or an epoxide group.

In some embodiments, the anchor region is a polystyrene bead, silica gelbead, polysaccharide bead, polyacrylamide bead, cellulose bead,polysaccharide, derivatized cellulose, polyacrylate, polyethyleneimine,polyacrylamide, UV-activatable reactive group or peptidoglycanderivative, or a combination thereof. In some embodiments, the anchorregion binds to a support material after a short period of UVirradiation.

In some embodiments, the enzyme-labile or enzyme-reactive region reactswith one or more target enzymes selected from elastase, lysozyme,myeloperoxidase, leukocyte peroxidase, esterase, lipase, napsin(aspartyl protease), glucosylceramidase glucuronidase, palmitoyl proteinthioesterase, cathepsins A, B, D, G, L, S, Z, Acid ceramidase,lactoferrin (LF), and proteinase-3,□-lactamase and other similarenzymes, or a combination thereof. In some embodiments, theenzyme-labile or enzyme-reactive region reacts with elastase. In someembodiments, the enzyme-labile or enzyme-reactive region reacts withlysozyme. In some embodiments, the enzyme-labile or enzyme-reactiveregion reacts with cathepsin G. In some embodiments, the enzyme-labileor enzyme-reactive region reacts with myeloperoxidase.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide, peptidomimetic, or protein that is labile toelastase or cathepsin G, or a combination thereof. In some embodiments,the enzyme-labile or enzyme-reactive region comprises a peptide,peptidomimetic, or protein that is labile to elastase. In someembodiments, the enzyme-labile or enzyme-reactive region comprises apeptide, peptidomimetic, or protein that is labile to cathepsin G.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide that is labile to elastase. In some embodiments, theenzyme-labile or enzyme-reactive region comprises a peptide comprisingan amino acid sequence of X_(y)AAP(V/F/A)X_(y)-L-Z, wherein each X isindependently any amino acid, each y is independently an integer greaterthan 0, or each y is independently an integer from 1 to 50, or each y isindependently an integer from 1 to 10, or each y is independently aninteger from 1 to 6, L is a linking moiety, and Z is a moiety capable ofcausing a visible color change or a detectable electronic change; andthe peptide is labile to elastase. In some embodiments, one or more ofthe amino acids in the amino acid sequence is protected. In someembodiments, one or more of the amino acids in the amino acid sequenceis protected with an fmoc group. In some embodiments, one of the aminoacid in the amino acid sequence is protected with an fmoc group.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence ofX_(y)AAP(V/F/A)X_(y)-Z, wherein each X is independently any amino acid,each y is independently an integer greater than 0, or each y isindependently an integer from 1 to 50, or each y is independently aninteger from 1 to 10, or each y is independently an integer from 1 to 6,and Z is a moiety capable of causing a visible color change or adetectable electronic change; and the peptide is labile to elastase. Insome embodiments, one or more of the amino acids in the amino acidsequence is protected. In some embodiments, one or more of the aminoacids in the amino acid sequence is protected with an fmoc group. Insome embodiments, one of the amino acid in the amino acid sequence isprotected with an fmoc group.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence ofX_(y)AAPX_(y)-L-Z, wherein each X is independently any amino acid, eachy is independently a number selected from 1 to 50, or each y isindependently an integer from 1 to 10, or each y is independently aninteger from 1 to 6, L is a linking moiety, and Z is a moiety capable ofcausing a visible color change or a detectable electronic change; andthe peptide is labile to elastase. In some embodiments, one or more ofthe amino acids in the amino acid sequence is protected. In someembodiments, one or more of the amino acids in the amino acid sequenceis protected with an fmoc group. In some embodiments, one of the aminoacid in the amino acid sequence is protected with an fmoc group.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence ofX_(y)AAPX_(y)-Z, wherein each X is independently any amino acid, each yis independently a number selected from 1 to 50, or each y isindependently an integer from 1 to 10, or each y is independently aninteger from 1 to 6, and Z is a moiety capable of causing a visiblecolor change or a detectable electronic change; and the peptide islabile to elastase. In some embodiments, one or more of the amino acidsin the amino acid sequence is protected. In some embodiments, one ormore of the amino acids in the amino acid sequence is protected with anfmoc group. In some embodiments, one of the amino acid in the amino acidsequence is protected with an fmoc group.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence ofX_(y)UUUU_(y)-Z, wherein each X is independently any amino acid, each yis independently a number selected from 1 to 50, or each y isindependently an integer from 1 to 10, or each y is independently aninteger from 1 to 6, U represents an amino acid selected from arginine,lysine, glycine or alanine, and Z is a moiety capable of causing avisible color change or a detectable electronic change

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence ofX_(y)UUUUUU_(y)-Z, wherein each X is independently any amino acid, eachy is independently a number selected from 1 to 50, or each y isindependently an integer from 1 to 10, or each y is independently aninteger from 1 to 6, U represents an amino acid selected from LEVLFQ,and Z is a moiety capable of causing a visible color change or adetectable electronic change

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide that is labile to cathepsin G.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence ofX_(y)N⁴N³N²N¹X_(y)-L-Z, wherein each X is independently any amino acid;each y is independently a number selected from 0 to 6; N⁴ is selectedfrom alanine, glycine, valine, and glutamine; N³ is selected fromalanine, glycine, proline, lysine, and serine: N² is selected fromproline, alanine, and glycine; N¹ is selected from serine, lysine,phenylalanine, arginine, leucine, and methionine; L is a linking moiety,and Z is a moiety capable of causing a visible color change or adetectable electronic change; and the peptide is labile to cathepsin G.In some embodiments, one or more of the amino acids in the amino acidsequence is protected. In some embodiments, one or more of the aminoacids in the amino acid sequence is protected with an fmoc group. Insome embodiments, one of the amino acid in the amino acid sequence isprotected with an fmoc group.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence ofX_(y)N⁴N³N²N¹X_(y)-Z, wherein each X is independently any amino acid;each y is independently a number selected from 0 to 6; N⁴ is selectedfrom alanine, glycine, valine, and glutamine; N³ is selected fromalanine, glycine, proline, lysine, and serine. N² is selected fromproline, alanine, and glycine; N¹ is selected from serine, lysine,phenylalanine, arginine, leucine, and methionine; and Z is a moietycapable of causing a visible color change or a detectable electronicchange; and the peptide is labile to cathepsin G. In some embodiments,one or more of the amino acids in the amino acid sequence is protected.In some embodiments, one or more of the amino acids in the amino acidsequence is protected with an fmoc group. In some embodiments, one ofthe amino acid in the amino acid sequence is protected with an fmocgroup.

In some embodiments, Z is a peroxidase substrate, an arylamine, an aminophenol, an aminophenyl ether, an indoxyl, a neutral dye, a charged dye,a nanoparticle, or a colloidal gold particle. In some embodiments, Z isa peroxidase substrate. In some embodiments, the peroxidase substrate isselected from p-aminophenol, ABTS(2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammoniumsalt), 3,3′-diaminobenzidine, DCPIP, N,N-dimethyl-p-phenylenediamine,o-dianisidine, p-phenylenediamine, 4-chloro-I-naphthol,o-phenylenediamine N-(4-aminobutyl)-N-ethylisoluminol,3-amino-9-ethylcarbazole, 4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), indoxyl, indigo,Fast Blue RR, 4-chloro-7-nitrobenzofurazan. In some embodiments, Z is anarylamine. In some embodiments, Z is an amino phenol. In someembodiments, Z is an aminophenol ether. In some embodiments, Z is anindoxyl. In some embodiments, Z is a neutral dye. In some embodiments, Zis a charged dye. In some embodiments, the charged dye is selected fromremazole brilliant blue, toluidine blue, reactive black 5, remazolbrilliant blue, reactive violet 5, and reactive orange 16, or ahydrolytic or ammonolytic derivatives thereof. In some embodiments, thecharged dye is remazole brilliant blue, or a hydrolytic or ammonolyticderivatives thereof. In some embodiments, the charged dye is toluidineblue. In some embodiments, the charged dye is reactive black 5, or ahydrolytic or ammonolytic derivatives thereof. In some embodiments, thecharged dye is reactive violet 5, or hydrolytic or ammonolyticderivatives thereof. In some embodiments, the charged dye is reactiveorange 16, or hydrolytic or ammonolytic derivatives thereof.

In some embodiments, Z is a dichlorotriazine-based reactive dye such asreactive blue 4, reactive red 120, reactive blue 2, reactive green 19and reactive brown 10. In some embodiments, the dichlorotriazine-basedreactive dye appears black.

In some embodiments, Z is a reactive dye containing asulfonylethyl-hydrogensulphate-reactive-group. In some embodiments, thereactive dye is reactive black 5, remazol brilliant blue, reactiveviolet 5 or reactive orange 16. In some embodiments, the reactive dye isreactive black 5. In some embodiments, the reactive dye is remazolbrilliant blue. In some embodiments, the reactive dye is reactive violet5. In some embodiments, the reactive dye is reactive orange 16. In someembodiments, the reactive dye is reactive black 5, remazol brilliantblue, or reactive violet 5. In some embodiments, the reactive dye isreactive black 5 or remazol brilliant blue.

In some embodiments, Z is a nanoparticle. In some embodiments, Z is acolloidal gold particle.

In some embodiments, Z is a charged dye, an indole derivative, or aluminol derivative. In some embodiments, Z is an indole derivative. Insome embodiments, Z is a luminol derivative.

In some embodiments, the enzyme-labile or enzyme-reactive region reactswith lysozyme. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises peptidoglycan.

In some embodiments, the enzyme-labile or enzyme-reactive region is abeta lactam.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a phenol, an amino phenol, an aminophenyl ether, an indoxyl,or a quinone. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises a phenol. In some embodiments, the enzyme-labile orenzyme-reactive region comprises an amino phenol. In some embodiments,the enzyme-labile or enzyme-reactive region comprises an amino phenolether. In some embodiments, the enzyme-label or enzyme-reactive regioncomprises an indoxyl. In some embodiments, the enzyme-labile orenzyme-reactive region comprises a quinone. In some embodiments, theenzyme-labile or enzyme-reactive region reacts with myeloperoxidase butdoes not react with heme.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peroxidase substrate, an arylamine, an amino phenol, aneutral dye, a charged dye, a nanoparticle, or a colloidal goldparticle. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises a peroxidase substrate. In some embodiments, theperoxidase substrate is selected from p-aminophenol, ABTS(2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt),3,3′-diaminobenzidine, DCPIP, N,N-dimethyl-p-phenylenediamine,o-dianisidine, p-phenylenediamine, 4-chloro-1-naphthol,o-phenylenediamine N-(4-aminobutyl)-N-ethylisoluminol,3-amino-9-ethylcarbazole, 4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and4-chloro-7-nitrobenzofurazan, Fast Blue RR, N-(2-hydroxy)tetradecyl-FastBlue RR. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises an arylamine. In some embodiments, the enzyme-labile orenzyme-reactive region comprises an amino phenol. In some embodiments,the enzyme-labile or enzyme-reactive region comprises a neutral dye. Insome embodiments, the enzyme-labile or enzyme-reactive region comprisesa charged dye. In some embodiments, the charged dye is selected fromremazole brilliant blue, toluidine blue, reactive black 5, remazolbrilliant blue, reactive violet 5, and reactive orange 16, or hydrolyticor ammonolytic derivatives of each of these. In some embodiments, thecharged dye is remazole brilliant blue, or hydrolytic or ammonolyticderivatives thereof. In some embodiments, the charged dye is toluidineblue. In some embodiments, the charged dye is reactive black 5, orhydrolytic or ammonolytic derivatives thereof. In some embodiments, thecharged dye is reactive violet 5, or hydrolytic or ammonolyticderivatives thereof. In some embodiments, the charged dye is reactiveorange 16, or hydrolytic or ammonolytic derivatives thereof.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a dichlorotriazine-based reactive dye such as reactive blue 4,reactive red 120, reactive blue 2, reactive green 19 and reactive brown10.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a nanoparticle. In some embodiments, Z is a colloidal goldparticle.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a charged dye, an indole derivative, or a luminol derivative.In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises an indole derivative. In some embodiments, the enzyme-labileor enzyme-reactive region comprises a luminol derivative.

In some embodiments, the indicator region comprises a dye that presentsa visible color change in normal ambient lighting. In some embodiments,the dye has a contrasting color to wound products, which are commonlyred, yellow, or brown. In further embodiments, the dye is violet, blueor dark green. In some embodiments, the dye is violet. In someembodiments, the dye is blue. In some embodiments, the dye is darkgreen. In some embodiments, the dye has low molecular weight, ischarged, contains reactive or linkable groups, is stable to gammairradiation, and is deeply colored. In some embodiments, the dye isselected from cibracron series dyes, azo dyes, and remazol dyes, orhydrolytic or ammonolytic derivatives thereof. In some embodiments, thedye is selected from cibracron series dyes. In some embodiments, the dyeis selected from azo dyes. In some embodiments, the dye is selected fromremazol dyes, or hydrolytic or ammonolytic derivatives thereof. In someembodiments, the dye is selected from rhodamine, coumarin, cyanine,xanthene, polymethine, pyrene, dipyrromethene borondifluoride,napthalimide, a phycobiliprotein, peridinium chlorophyll proteins,fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594,tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6F,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.54, Cy7®, Cy-Chrome, DyLight350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633,DyLight 649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP(peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6-)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350,Alex Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-aceticacid, BODIPY® FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568,BODIPY® 630/650, BODIPY® 650/665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR,and dimethylaminoazobenzenesulfonic acid (dabsyl), or conjugatesthereof, or combinations thereof.

In some embodiments, the indicator region comprises adichlorotriazine-based reactive dye such as reactive blue 4, reactivered 120, reactive blue 2, reactive green 19 and reactive brown 10. Insome embodiments, the dichlorotriazine-based reactive dye appears black.

In some embodiments, the indicator region comprises the reaction productof a reactive dye containing asulfonylethyl-hydrogensulphate-reactive-group. In some embodiments, thereactive dye is reactive black 5, remazol brilliant blue, reactiveviolet 5 or reactive orange 16. In some embodiments, the reactive dye isreactive black 5. In some embodiments, the reactive dye is remazolbrilliant blue. In some embodiments, the reactive dye is reactive violet5. In some embodiments, the reactive dye is reactive orange 16. In someembodiments, the reactive dye is reactive black 5, remazol brilliantblue, or reactive violet 5. In some embodiments, the reactive dye isreactive black 5 or remazol brilliant blue.

In some embodiments, the indicator region comprises a particle (e.g.,colloidal metal or quantum dots) that present color changes in normalambient lighting. In some embodiments, the indicator region comprises ananoparticle. In some embodiments, the indicator region comprises acolloidal gold particle.

In some embodiments, the indicator region comprises a dye that presentsa visible color change under UV light. In some embodiments, theindicator region comprises a dye that is fluorescent. In someembodiments, the indicator region comprises a dye that is luminescent.

In some embodiments, the indicator region comprises an enzyme-reactivemoiety. In some embodiments, the enzyme-reactive moiety interacts withan accessory enzyme to produce a product that is visible to the nakedeye or detectable by electronic means. In some embodiments, theenzyme-reactive moiety interacts with an accessory enzyme to produce aproduct that is visible to the naked eye. In some embodiments, theenzyme-reactive moiety interacts with an accessory enzyme to produce aproduct that is detectable by electronic means. In some embodiments, theindicator region comprises an indoxyl glycoside or galactoside that iscleaved by glucuronidase, glucosidase or galactosidase depending on theterminal sugar used, to produce indigo. In some embodiments, theindicator region comprises a phenol or napthol that is oxidized by anaccessory enzyme to produce a visible product. In some embodiments, theindicator region comprises a phenol that is oxidized by peroxidase toproduce a visible product. In some embodiments, the indicator regioncomprises a metallo motif that is detectable by electronic means. Insome embodiments, the indicator region comprises a ferrocene orferrocene analog that is detectable by electronic means. In someembodiments, the accessory enzyme is selected from lipase, esterase,peroxidase, oxidase, glycosidase, and glucosidase. In some embodiments,the accessory enzyme is not present in the wound fluid. In someembodiments, the accessory enzyme is present in the wound fluid. In someembodiments, the enzyme-reactive moiety interacts with an accessoryenzyme to produce a product that is visible under UV light.

In some embodiments, the chemical entity consists essentially of atleast one anchor region, at least one enzyme-labile or enzyme-reactiveregion, and at least one indicator region. In some embodiments, thechemical entity consists essentially of at least one enzyme-labile orenzyme-reactive region, and at least one indicator region. In someembodiments, the chemical entity consists essentially of at least oneanchor region and at least one enzyme-labile or enzyme-reactive region.In some embodiments, the chemical entity is capable of being bound to asupport material without an anchor region.

In some embodiments, the chemical entity is printed on or in a supportmaterial such as filter paper or a woven or non-woven material that iscapable of being wet by a wound fluid and which displays capillaryaction. In some embodiments, the reporting entity or chemical entity ischemically bonded onto or into a support material such as filter paperor a woven or non-woven material that is capable of being wet by a woundfluid and which displays capillary action that is similar in alldimensions. In some embodiments, the chemical entity is ionically boundonto or into a support material such as filter paper or a woven ornon-woven material that is capable of being wet by a wound fluid andwhich displays capillary action. In some embodiments, the chemicalentity is covalently bound onto or into a support material such asfilter paper or a woven or non-woven material that is capable of beingwet by a wound fluid and which displays capillary action. Supportmaterial includes, but is not limited to, cellulose, polyamide,polyester, polyacrylate and other similar polymers that are useful asfibers. In some embodiments, the support material is cellulose. In someembodiments, the support material is polyamide. In some embodiments, thesupport material is polyester. In some embodiments, the support materialis polyacrylate.

In some instances, the pH of a wound can influence many factors of woundhealing, such as angiogenesis, protease activity, oxygen release, andbacterial toxicity. Chronic nonhealing wounds may have an elevatedalkaline environment. As the wound progresses towards healing, the pH ofthe wound moves to neutral and then becomes acidic. Monitoring of the pHof the wound may provide a method to assess the condition of the wound(e.g., infection or no infection) and aid in determining a wound'sresponse to treatment.

In some embodiments, the pH-sensitive moiety is bromothymol blue, phenolred, bromophenol red, chlorophenol red, thymol blue, bromocresol green,bromocresol purple; or other sulfophthalein dyes.

In some embodiments, the MPO responsive indicator incorporates an alkylaniline amide with a molecular weight greater than 459 Da. In preferredembodiments, this indicator has the following structure:

-   -   Wherein,    -   R¹=—CH₃, —CH₂CH₃; —CH₂CH₂CH₂CH₃    -   X=—C(=0)-, —S(=0)₂-    -   R²=can be but is not limited to:    -   —Ci-Cio alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl,        alkylheteroaryl    -   wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl and        alkylheteroaryl groups are optionally substituted by one to five        substituents selected independently from: ferrocene, halogen (as        can be F, Cl, Br, I), (C₁-C₄)alkyl, (C₁-C₄)alkenyl,        (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl,        (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, —NR⁴R⁵,        R⁴C(=0)-, R⁴C(=0)0-, R⁴OC(=0)0-, R⁴ HC(=0)-, R⁴C(=0) H—,        R⁴R⁵NC(=0)-, R⁴OC(=0)-    -   R⁴ and R⁵ can independently be, but are not limited to:        -   (C₁-Ci₂)alkyl        -   (C₁-Ci₂)alkenyl        -   (C₁-Ci₂)akynyl        -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl        -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl        -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl        -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   wherein alkyl, alkenyl, alkynyl, aryl and heteroaryl are        optionally substituted by one to five substituents selected        independently from halogen (as can be F, Cl, Br, I),        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-Ci₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy,    -   or N(R⁴R⁵) is an aziidine, azetidine, pyrrolidine, piped dine,        azepane or azocane, 1-substituted piperazine, or morpholine        moiety    -   Y=—NH—CH2-CH(OH)—,—NH—, —NH—C(═O)—NH—, —NH—C(═S)—NH—    -   R³ can be but is not limited to:    -   —C6-C30 alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl,        alkylheteroaryl    -   wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl and        alkylheteroaryl groups are optionally substituted by one to five        substituents selected independently from: ferrocene, halogen (as        can be F, Cl, Br, I), (C₁-C₄)alkyl, (C₁-C₄)alkenyl,        (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl,        (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, cyano (—CN),        azido (—N₃), —NR⁴R⁵, R⁴C(═O)—, R⁴C(═O)O—, R⁴OC(═O)O—,        R⁴NHC(═O)—, R⁴C(═O) H—, R⁴R⁵NC(═O)—, R⁴OC(═O)—

In some embodiments, it is desired that the indicator substrates stay inplace and react in place. Thus, limited solubility in aqueous systems ispreferred. The ability to stay in place on a solid phase is defined asthe water resistance and the means by which it is measured is recordedin example 114. In preferred embodiments, the water resistance of asubstrate is greater than one, and in still more preferred embodiments,it is greater than 2.

In some embodiments, disclosed herein are compounds of the followingformula (Formula D):

-   -   wherein,    -   R¹=—CH₃, —CH₂CH₃; —CH₂CH₂CH₂CH₃    -   X=—C(═O)—, —S(═O)₂—    -   R²=can be but is not limited to: —C₁-C₁₀ alkyl, alkenyl,        alkynyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl wherein        alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl and        alkylheteroaryl groups are optionally substituted by one to five        substituents selected independently from: ferrocene, halogen (as        can be F, Cl, Br, I), (C₁-C₄)alkyl, (C₁-C₄)alkenyl,        (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl,        (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, —NR⁴R⁵,        R⁴C(═O)—, R⁴C(═O)O—, R⁴OC(═O)O—, R⁴NHC(═O)—, R⁴C(═O)NH—,        R⁴R⁵NC(═O)—, R⁴OC(═O)—    -   R4 and R⁵ can independently be, but are not limited to:        —(C₁-C₁₂)alkyl—(C₁-C₁₂)alkenyl—(C₁-C₁₂)akynyl—(C₁-C₈)[(C₁-C₄)alkoxy]alkyl—(C₁-C₈)[(C₁-C₄)alkoxy]alkenyl—(C₆-C₁₀)aryl-(C₁-C₅)alkyl—(C₂-C₉)heteroaryl-(C₁-C₅)alkyl;        wherein alkyl, alkenyl, alkynyl, aryl and heteroaryl are        optionally substituted by one to five substituents selected        independently from halogen (as can be F, Cl, Br, I),        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-Ci₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, or N(R⁴R⁵) is an aziidine, azetidine,        pyrrolidine, piped dine, azepane or azocane, 1-substituted        piperazine, or morpholine moiety; Y=—NH—CH2-CH(OH)—,—NH—,        —NH—C(═O)—NH—, —NH—C(═S)—NH—    -   R³ can be but is not limited to: —C₆-C₃₀ alkyl, alkenyl,        alkynyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, wherein        alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl and        alkylheteroaryl groups are optionally substituted by one to five        substituents selected independently from: ferrocene, halogen (as        can be F, Cl, Br, I), (C₁-C₄)alkyl, (C₁-C₄)alkenyl,        (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl,        (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, cyano (—CN),        azido (—N₃), —R⁴R⁵, R⁴C(═O)—, R⁴C(═O)O—, R⁴OC(═O)O—, R4 HC(═O)—,        R⁴C(═O) H—, R⁴R⁵NC(═O)—, R⁴OC(═O)—.

In some embodiments, disclosed herein are compounds of the followingformula (Formula A)

-   -   wherein    -   Y is O, or N    -   Ar is

-   -   or ferrocene;    -   R is a —C₁-C₁₀ alkyl, alkenyl, alkynyl, aryl, heteroaryl,        alkylaiyl, or alkylheteroaryl;    -   α is A, V, F, G, M, R, or L;    -   P is a bond, or independently, P, F, A, R, L, or G;    -   γ is a bond, or independently, P, A, R, L, or G;    -   δ is a bond, or independently, P, A, R, L, G, or V;    -   ε is a bond, or independently, P, A, R, L, G, V, or E;    -   φ is a bond, or independently, P, A, R, L, or G;    -   wherein, if β, γ, δ, ε, and φ, are each bonds, then the        N-terminal of α is bonded to X;    -   X is a peptide chain comprising from 0 to 14 amino acids;    -   PG is —C(═O)—O—R¹ wherein R¹ is C₁-C₃₀ alkyl, t-butyl, or        methylfluorenyl, or —C(═O)—R² wherein R² is —C₁-C₃₀ alkyl,        alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, or        alkylheteroaryl.

In some embodiments, disclosed herein are compounds of the followingformula (Formula B)

-   -   wherein    -   L is A, AA, V, or triazolyl,    -   Z is ferrocene, crystal violet, malachite green, toluidine blue,        reactive black 5, remazol brilliant blue, reactive violet 5, and        reactive orange 16, reactive blue 4, reactive red 120, reactive        blue 2, reactive green 19, or reactive brown 10    -   α is selected from: A, V, F, G, M, R, or L    -   β is a bond, or independently, P, F, A, R, L, or G    -   γ is a bond, or independently, P, A, R, L, or G    -   δ is a bond, or independently, P, A, R, L, G, or V    -   ε is a bond, or independently, P, A, R, L, G, V, or E    -   φ is a bond, or independently, P, A, R, L, or G,    -   wherein, if β, γ, δ, ε, and φ, are each bonds, then the        N-terminal of a is bonded to X;    -   X is a peptide chain comprising from 0 to 14 AA, wherein AA is        an amino acid, a polyamine, or a polyoxyalkylene;    -   PG is polystyrene bead, silica gel bead, polysaccharide bead,        polyacrylamide bead, cellulose bead, polysaccharide, derivatized        cellulose, polyacrylate, polyethyleneimine, polyacrylamide,        UV-activatable group, phenolic azide, epoxide, peptidoglycan, an        aliphatic chain, an aliphatic alcohol chain, an aliphatic amine,        mercaptoethyl, a multi-cyclic or multi-aromatic ring system, a        lipophilic group, or a combination thereof, —C(═O)—O—R¹ wherein        R¹ is C₁-C₃₀ alkyl, t-butyl, methylfluorenyl, —C(═O)—R² wherein        R² is —C₁-C₃₀ alkyl, alkenyl, alkynyl, aryl, heteroaryl,        alkylaryl, or alkylheteroaryl.

In general, the elastase substrates have the formula a-b-c-d-e-f,wherein

In one embodiment,

-   -   a is selected from: A, V, F, G, M, R, L    -   b is selected from: no amino acid, or independently, P, F, A, R,        L, G    -   g is selected from: no amino acid, or independently, P, A, R, L,        G    -   d is selected from: no amino acid, or independently, P, A, R, L,        G, V    -   e is selected from: no amino acid, or independently, P, A, R, L,        G, V, E    -   f is selected from: no amino acid, or independently, P, A, R, L,        G.

In another embodiment, elastase substrates have the formula a-b-c-d-e-f,wherein

-   -   a is selected from: A, V, F, G, M, R, L    -   b is selected from: no amino acid, or independently, P, F, A, G    -   g is selected from: no amino acid, or independently, P, A, R, L,        G    -   d is selected from: no amino acid, or independently, P, A, R, L,        G, V    -   e is selected from: no amino acid, or independently, P, A, G, V,        E    -   f is selected from: no amino acid, or independently, P, A, G.

In another embodiment, elastase substrates have the formula a-b-c-d-e-f,wherein

-   -   a is selected from: A, V, F, G, M, R, L    -   b is selected from: no amino acid, or independently, P, F, A, G    -   g is selected from: no amino acid, or independently, P, A, L, G    -   d is selected from: no amino acid, or independently, P, A, L, G,        V    -   e is selected from: no amino acid, or independently, P, A, G, V,        E    -   f is selected from: no amino acid, or independently, P, A, G.

In another embodiment, elastase substrates have the formula a-b-c-d-e-f,wherein

-   -   a is selected from: A, V, F, G    -   b is selected from: no amino acid, or independently, P, F, A, G    -   g is selected from: no amino acid, or independently, P, A, G    -   d is selected from: no amino acid, or independently, P, A, G,    -   e is selected from: no amino acid, or independently, A, G, V,    -   f is selected from: no amino acid, or independently, A, G.

Especially, in another embodiment, elastase substrates have the formulaa-b-c-d-e-f, wherein

-   -   a is selected from: A, V, F    -   b is selected from: no amino acid, or independently, P, F, A,    -   g is selected from: no amino acid, or independently, A, G    -   d is selected from: no amino acid, or independently, A, G,    -   e is selected from: no amino acid, or independently, A, G,    -   f is selected from: no amino acid, or independently, A, G.

Preferably, elastase substrates have the formula a-b-c-d-e-f, wherein

-   -   a is selected from: A, V, F    -   b is selected from: no amino acid, or independently, P, F, A,    -   g is selected from: no amino acid, or independently, A,    -   d is selected from: no amino acid, or independently, A,    -   e is selected from: no amino acid, or independently, A,    -   f is selected from: no amino acid, or independently, A.

DESCRIPTION OF THE FIGURES

FIG. 1 : Exemplary embodiment of compounds disclosed herein.

FIG. 2 : Exemplary embodiment of compounds disclosed herein withmultiple reaction requirement toward MPO and elastase (e.g. example 9,Methyl-3-Fmoc-AAPV-amide-4-aminophenol orMethyl-3-amino-4-Fmoc-AAPV-amidebenzoate) [SEQ ID NO: 2].

FIG. 3A: Exemplary embodiment of compounds disclosed herein withmultiple reaction requirement toward elastase.

FIG. 3B: Exemplary embodiment of compounds disclosed herein withmultiple reaction requirement toward elastase.

FIG. 4A: Exemplary embodiment of compounds disclosed herein withreaction toward elastase in which overlapping function for the anchor isshown for an elastase substrate.

FIG. 4B: Exemplary embodiment of compounds disclosed herein withreaction toward MPO in which overlapping function for an indicator andreactive region is shown for an MPO substrate.

FIG. 5 : Synthetic routes toward p-lactamase substrates containing anindoxyl ether.

FIG. 6 : Esterase and lipase substrates containing an indoxylchromophore.

FIG. 7A: X-Gal, X-Gluc peptidoglycan and chitosan adducts formed via themethods of examples 103 and 104.

FIG. 7B: X-Man, peptidoglycan and chitosan adducts formed via themethods of examples 103 and 104.

FIG. 7C: Esterase substrates containing an indoxyl chromophore coupledto Peptidoglycan, chitosan or a peptide via the Suzuki product ofexample 103.

FIG. 8 : A scheme for the carboxymethylation of peptidoglycan andsubsequent derivatisation.

FIG. 9A: A tube containing an indicator substrate for use in detectinginfection in tissues or body fluids emanating from an organ of interest(airway, blood, urine etc.). The tube is a PVC material into which anonwoven printed with the substrate from example 18 has been inserted.

FIG. 9B: A tubing insert designed to accommodate a flat filter paperseries containing one or more substrates such as those from examples 10or 18 with that of example 95 serving as a positive control (center).

FIG. 9C: Tubing containing a polyolefin inner cylinder serving as thecarrier for the substrate of example 11. This material serves as thelining in the hollow cylinder (right).

FIG. 9D: Adaptation of the hollow lining to the PVC tubing in exemplaryform.

FIG. 10A: A stick containing indicator substrates from examples 10 and18 can be used to detect the reaction of fluids derived from thenegative pressure treatment of wounds.

FIG. 10B: The same stick format can be used to monitor the aspirates ofthe airway or sputum to detect incipient infection. In this image thecalibration of the sticks for the response to elastase or MPO isindicated.

FIG. 11 : A scheme for the production of a carboxy ferrocenepeptidoglycan conjugate

FIG. 12 : The sulfate salt the product of example 10 and Fast Blue RR.

FIG. 13 : An example of the reaction product of MPO and the product ofexample 10 and Fast Blue RR.

FIG. 14 : A graph depicting biomarker response data for patientsclassified as “uninfected” who were sampled as an infection wasdeveloping.

DETAILED DESCRIPTION

Various aspects now will be described more fully hereinafter. Suchaspects may, however, be embodied in many different forms and should notbe construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey its scope to those skilled in theart.

Throughout this disclosure, various patents, patent applications andpublications are referenced. The disclosures of these patents, patentapplications and publications in their entireties are incorporated intothis disclosure by reference in order to more fully describe the stateof the art as known to those skilled therein as of the date of thisdisclosure. This disclosure will govern in the instance that there isany inconsistency between the patents, patent applications andpublications cited and this disclosure.

I. Definitions

Where a range of values is provided, it is intended that eachintervening value between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. For example, if a range of 1 μm to 8μm is stated, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μmare also explicitly disclosed, as well as the range of values greaterthan or equal to 1 μm and the range of values less than or equal to 8μm.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference toa “polymer” includes a single polymer as well as two or more of the sameor different polymers, reference to an “excipient” includes a singleexcipient as well as two or more of the same or different excipients,and the like.

The word “about” when immediately preceding a numerical value means arange of plus or minus 10% of that value, e.g., “about 50” means 45 to55, “about 25,000” means 22,500 to 27,500, etc., unless the context ofthe disclosure indicates otherwise, or is inconsistent with such aninterpretation. For example in a list of numerical values such as “about49, about 50, about 55, “about 50” means a range extending to less thanhalf the interval(s) between the preceding and subsequent values, e.g.,more than 49.5 to less than 52.5. Furthermore, the phrases “less thanabout” a value or “greater than about” a value should be understood inview of the definition of the term “about” provided herein.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 80%-95% or greater of some given quantity, e.g., at least 85%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.9%, or more % byweight or volume or any other parameter being measured. “Substantiallyfree” means nearly totally or completely absent of some given quantitysuch as being present at a level of less than about 1% to about 20% ofsome given quantity, e.g., less than 10%, less than 9%, less than 8%,less than 7%), less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, less than 1%, less than 0.5%), less than 0.1%, or less %by weight or volume or any other parameter being measured. In someembodiments, “substantially free” means presence at a level of less thanor equal to 1-5% by weight of the pharmaceutical composition.

II. Overview

Provided herein are compositions and systems for the therapy anddiagnosis of wounds and wound management, wherein the compositions, whenin use, indicate the presence of elevated enzyme levels in a wound insitu.

As used herein, a “wound” refers to physical disruption of thecontinuity or integrity of tissue structure. “Wound healing” refers tothe restoration of tissue integrity. It will be understood that this canrefer to a partial or a full restoration of tissue integrity. Treatmentof a wound thus refers to the promotion, improvement, progression,acceleration, or otherwise advancement of one or more stages orprocesses associated with the wound healing process.

The wound may be acute or chronic. Chronic wounds, including pressuresores, venous leg ulcers and diabetic foot ulcers, can simply bedescribed as wounds that fail to heal. Whilst the exact molecularpathogenesis of chronic wounds is not fully understood, it isacknowledged to be multi-factorial. As the normal responses of residentand migratory cells during acute injury become impaired, these woundsare characterized by a prolonged inflammatory response, defective woundextracellular matrix (ECM) remodeling and a failure ofre-epithelialization.

The wound may be any internal wound, e.g., where the external structuralintegrity of the skin is maintained, such as in bruising or internalulceration, or external wounds, particularly cutaneous wounds, andconsequently the tissue may be any internal or external bodily tissue.In one embodiment the tissue is skin (such as human skin), i.e. thewound is a cutaneous wound, such as a dermal or epidermal wound.

The human skin is composed of two distinct layers, the epidermis and thedermis, below which lies the subcutaneous tissue. The primary functionsof the skin are to provide protection to the internal organs and tissuesfrom external trauma and pathogenic infection, sensation andthermoregulation. The skin tissue of most mammals is structuredsimilarly.

The outermost layer of skin, the epidermis, is approximately 0.04 mmthick, is avascular, is comprised of four cell types (keratinocytes,melanocytes, Langertians cells, and Merkel cells), and is stratifiedinto several epithelial cell layers. The inner-most epithelial layer ofthe epidermis is the basement membrane, which is in direct contact with,and anchors the epidermis to, the dermis. All epithelial cell divisionoccurring in skin takes place at the basement membrane. After celldivision, the epithelial cells migrate towards the outer surface of theepidermis. During this migration, the cells undergo a process known askeratinization, whereby nuclei are lost and the cells are transformedinto tough, flat, resistant non-living cells. Migration is completedwhen the cells reach the outermost epidermal structure, the stratumcorneum, a dry, waterproof squamous cell layer which helps to preventdesiccation of the underlying tissue. This layer of dead epithelialcells is continuously being sloughed off and replaced by keratinizedcells moving to the surface from the basement membrane. Because theepidermal epithelium is avascular, the basement membrane is dependentupon the dermis for its nutrient supply.

The dermis is a highly vascularized tissue layer supplying nutrients tothe epidermis. In addition, the dermis contains nerve endings,lymphatics, collagen protein, and connective tissue. The dermis isapproximately 0.5 mm thick and is composed predominantly of fibroblastsand macrophages. These cell types are largely responsible for theproduction and maintenance of collagen, the protein found in all animalconnective tissue, including the skin. Collagen is primarily responsiblefor the skin's resilient, elastic nature. The subcutaneous tissue, foundbeneath the collagen-rich dermis, provides for skin mobility,insulation, calorie storage, and blood to the tissues above it.

Wounds can be classified in one of two general categories, partialthickness wounds or full thickness wounds. A partial thickness wound islimited to the epidermis and superficial dermis with no damage to thedermal blood vessels. A full thickness wound involves disruption of thedermis and extends to deeper tissue layers, involving disruption of thedermal blood vessels. The healing of the partial thickness wound occursby simple regeneration of epithelial tissue. Wound healing in fullthickness wounds is more complex. Cutaneous wounds contemplated hereinmay be either partial thickness or full thickness wounds.

Wounds contemplated herein include cuts and lacerations, surgicalincisions or wounds, punctures, grazes, scratches, compression wounds,abrasions, friction wounds (e.g., nappy rash, friction blisters),decubitus ulcers (e.g., pressure or bed sores); thermal effect wounds(burns from cold and heat sources, either directly or throughconduction, convection, or radiation, and electrical sources), chemicalwounds (e.g. acid or alkali burns) or pathogenic infections (e.g.,viral, bacterial or fungal) including open or intact boils, skineruptions, blemishes and acne, ulcers, chronic wounds, (includingdiabetic-associated wounds such as lower leg and foot ulcers, venous legulcers and pressure sores), skin graft/transplant donor and recipientsites, immune response conditions, e.g., psoriasis and eczema, stomachor intestinal ulcers, oral wounds, including a ulcers of the mouth,damaged cartilage or bone, amputation wounds and corneal lesions.

Chemical Entities and Compositions Thereof

Embodiments described herein provide chemical entities, which may beused to diagnose and/or treat chronic wounds. The chemical entities andcompositions thereof, as described herein, are used in methods to detectthe level of one or more enzymes in a mammalian wound. In someembodiments, the chemical entities and compositions thereof, asdescribed herein, are used in methods to diagnose a chronic wound in amammal. In some embodiments, the chemical entities and compositionsthereof described herein are used in methods to diagnose an infectedwound in a mammal. In other embodiments, the chemical entities andcompositions thereof described herein are used in methods to treat awound in a mammal. In further embodiments, the chemical entities andcompositions thereof described herein are used in methods to treat aninfected or a chronic wound in a mammal.

In other embodiments, the chemical entities and compositions thereofdescribed herein may be used to diagnose one or more pathogenicmicroorganisms, e.g., bacteria or viruses, based on the detection of oneor more biomarkers that are specific for the microorganisms.

In one embodiment, provided herein is a chemical entity capable ofdetecting enzyme activity from a body fluid, the chemical entitycomprising: an anchor region (A); an enzyme-recognition site (R) and anindicator region (I). Under this embodiment, the chemical entity has abasic chemical structure A-R—I (Formula I), wherein A is an anchorregion; R is an enzyme recognition site and I is an indicator region.

In some embodiments, the anchor region (A) is associated with theindicator region (I) via an enzyme recognition site (R). Under thisembodiment, the enzyme recognition site is a structure or a motif thatallows binding to an enzyme.

In one embodiment, the enzyme recognition site (R) is naturally presentin the anchor region. Cleavage of the backbone of the anchor region byenzymes results in the mobilisation of fragments, which in turn maybecome substrates for the enzyme.

In another embodiment, the enzyme recognition site (R) is introduced inthe anchor region (A) via chemical modification. Alternately, the enzymerecognition site (R) may be naturally present in the indicator region(I) or synthetically introduced in the indicator region (I) via one ormore chemical modifications.

In one embodiment, the chemical entity of Formula I comprises an anchor(A) which is associated with the indicator (I), either covalently ornon-covalently, in which case, the recognition site (R) may be locatedin the associating moiety (e.g., via a covalent bond). As is understoodin the art, covalent bonds involve sharing of electrons between thebonded atoms. In contrast, non-covalent bonds may include, for example,ionic interactions, electrostatic interactions, hydrogen bondinginteractions, physiochemical interactions, van der Waal forces,Lewis-acid/Lewis-base interactions, or combinations thereof.

In one embodiment, the anchor A is associated with the indicator I via acovalent interaction to form the recognition site R. In anotherembodiment, the anchor A is associated with the indicator I via acovalent interaction that is not a part of the recognition site R. Underthe second embodiment, the R may wholly constitute a part of theindicator molecule or constitute a separate motif or moiety to which theindicator region is associated.

In some embodiments, the chemical entity further comprises anenzyme-labile or enzyme-reactive region.

In one embodiment, the recognition site (R) interacts with one or moretarget enzymes selected from the group consisting of selected fromlipase, esterase, peroxidase, oxidase, glycosidase, glucuronidase,glucosidase, galactosidase, and a combination thereof. In someembodiments, the enzyme-labile or enzyme-reactive region interacts withone or more target enzymes selected from Napsin (aspartyl protease),Glucosylceramidase glucuronidase, palmitoyl protein thioesterase,Cathepsins A, B, D, G, L, S, Z; acid ceramidase, lactoferrin (LF),lysozyme, myeloperoxidase (MPO), elastase, cathepsins, and proteinase-3elastase, lysozyme, esterase, lipase and a combination thereof.

Anchor Region (A)

In some embodiments of the chemical entity of Formula I, the anchorregion comprises a compound which is a polysaccharide, a cellulose, apolyacrylate, a polyethyleneimine, a polyacrylamide, a peptidoglycan, ora chitosan, or a monomer thereof, an oligomer thereof, a derivativethereof, a mixture or a combination thereof.

In one embodiment, the anchor A comprises a polysaccharide selected fromhydroxypropyl methylcellulose (UPMC), hydroxyethyl cellulose,hydroxymethyl cellulose, D-galactopyranoside, or a derivative thereof.

In one embodiment, the anchor A comprises peptidoglycan or a monomerthereof, an oligomer thereof, a derivative thereof, a mixture or acombination thereof. As is understood in the art, peptidoglycan, alsoknown as murein, is a polymer consisting of sugars and amino acids thatforms a mesh-like layer outside the plasma membrane of most bacteria,forming the cell wall, e.g., of a bacterium. The sugar componentconsists of alternating residues of β-(1,4) linked N-acetylglucosamineand N-acetylmuramic acid. Attached to the N-acetylmuramic acid is apeptide chain of three to five amino acids. The peptide chain can becross-linked to the peptide chain of another strand forming the 3Dmesh-like layer.

Accordingly, in one embodiment, the peptidoglycan may comprise at least2, at least 3, at least 4, at least 5, or more units of alternatingamino sugars selected from N-acetylglucosamine (GlcNAc or NAG) andN-acetylmuramic acid (MurNAc or NAM) or a combination thereof.Peptidoglycan, including, shorter fragments thereof, may be generatedfrom natural or synthetic sources. See, Lee et al., Chembiochem.,11(18):2525-9, 2010.

In another embodiment, the anchor A comprises a peptidoglycan or apolysaccharide derivative. Example peptidoglycan include peptidoglycansalts, water-soluble peptidoglycan, carboxylated peptidoglycan, etc.Representative examples of such derivatives include, e.g.,peptidoglycans containing carboxymethyl, carboxyethyl, carboxypropylgroup(s), optionally comprising one or more halogen, alcohol, ester oramide groups. Other particular examples include, halogenatedpeptidoglycans comprising one or more chlorine groups. Solublepeptidoglycans and fractions containing soluble fragments ofpeptidoglycans originating from the bacterium or bacterial wall ofNocardiae have been described in U.S. Pat. No. 5,017,359.

Polysaccharide derivatives include, e.g., hydroxyethyl cellulose (HEC),hydroxypropyl cellulose (HPC), ethyl hydroxyethyl cellulose (EHEC),carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose (CMHEC),hydroxypropyl hydroxyethyl cellulose (HPHEC), methyl cellulose (MC),methyl hydroxypropyl cellulose (MHPC), methyl hydroxyethyl cellulose(MHEC), carboxymethyl cellulose (CMC), hydroxypropyl methylcelluloseacetate succinate (HPMC-AS), hydrophobically modified hydroxyethylcellulose (hmHEC), hydrophobically modified hydroxypropyl cellulose(hmHPC), hydrophobically modified ethyl hydroxyethyl cellulose (hmEHEC),hydrophobically modified carboxymethyl hydroxyethyl cellulose (hmCMHEC),hydrophobically modified hydroxypropyl hydroxyethyl cellulose (hmHPHEC),hydrophobically modified methyl cellulose (hmMC), hydrophobicallymodified methyl hydroxypropyl cellulose (hmMHPC), hydrophobicallymodified methyl hydroxyethyl cellulose (hmMHEC), hydrophobicallymodified carboxymethyl methyl cellulose (hmCMMC), sulfoethyl cellulose(SEC), hydroxyethyl sulfoethyl cellulose (HESEC), hydroxypropylsulfoethyl cellulose (HPSEC), methyl hydroxyethyl sulfoethylcellulose(MHESEC), methyl hydroxypropyl sulfoethyl cellulose (MHPSEC),hydroxyethyl hydroxypropyl sulfoethyl cellulose (HEHPSEC), carboxymethylsulfoethyl cellulose (CMSEC), hydrophobically modified sulfoethylcellulose (hmSEC), hydrophobically modified hydroxyethyl sulfoethylcellulose (hmHESEC), hydrophobically modified hydroxypropyl sulfoethylcellulose (hmHPSEC) or hydrophobically modified hydroxyethylhydroxypropyl sulfoethyl cellulose (hmHEHPSEC). Particularly preferredcellulose derivatives are cellulose ethers having a thermal flocculationpoint in water, such as, for example, methyl cellulose, methylhydroxyethyl cellulose, methyl hydroxypropyl cellulose and hydroxypropylcellulose. See, U.S. Pat. No. 8,465,586.

In one embodiment, “peptidoglycan derivative” and “polysaccharidederivative” as used herein includes salts, amides, esters, enol ethers,enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals,acids, bases, solvates, hydrates or prodrugs of the peptidoglycan orpolysaccharide. Such derivatives may be readily prepared by those ofskill in this art using known methods for such derivatization. Incertain embodiments, the derivatives may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

In another embodiment, the derivative is a salt of the peptidoglycan orpolysaccharide compound, e.g., salts of Li, Na, K, Rb, Mg, Ca, Sr, orBa, preferably Na⁺, K⁺, Mg²⁺, Ca²⁺. Salts of charged peptidoglycan orpolysaccharides, such as sodium or calcium salts, are included by thisdefinition.

In some embodiments, the derivative anchor compound is a halogenatedanchor compound, e.g., halogenated polysaccharide, halogenatedpeptidoglycan, halogenated polyacrylate, halogenated polyethyleneimine,halogenated polyacrylamide, halogenated peptidoglycan, or halogenatedchitosan, or a monomer thereof, e.g., halogenated N-acetylglucosamine(GlcNAc or NAG) and/or halogenated N-acetylmuramic acid (MurNAc or NAM).The halogen is selected from the group consisting of Cl, Br, I;particularly, the halogen is Cl.

In some embodiments, the derivative compound is an isomer of the anchorcompound, term “isomer” includes compounds with the same formula but adifferent arrangement of atoms in the molecule. In embodiments, isomersof the compounds are “tautomers” or “stereoisomers” of the compounds.The term “stereoisomer” refers to compounds that differ in the chiralityof one or more stereocenters. Stereoisomers include enantiomers anddiastereomers. The term “tautomer” refers to alternate forms of acompound that differ in the position of a proton, such as enol-keto andimine-enamine tautomers, or the tautomeric forms of the anchor compound.

In some embodiments, the anchor compound may contain a combination ormixture of one or more of the aforementioned compounds. The term“combination” includes compounds containing more than one component,which may be conjugated or non-conjugated to one another. In oneembodiment, the anchor compound comprises a combination of one or moreof the aforementioned compounds which are conjugated to each other,e.g., via covalent or non-covalent interaction. As a particular example,the anchor may comprise a combination of chitosan and peptidoglycan.See, U.S. Patent Application Publication No. 2007/0167400. In anotherembodiment, the mixture may comprise commercially available preparationof peptidoglycan and polysaccharide mixture (PG/PS; Lee Labs Inc.,Grayson, GA; see, U.S. Pat. No. 8,129,518).

In some embodiments, the compounds include mixtures of theaforementioned polymeric compounds. The term “mixture” refers to amingling together of two or more substances without the occurrence of areaction by which they would lose their individual properties. Forinstance, a mixture of compound A and compound B may contain any weightratio of compound A and compound B, such that the total weight of themixture would amount to 100%, e.g., 99:1 weight ratio of compoundA/compound B or 1:99 weight ratio of compound A/compound B. A typicalmixture may contain about 2, 3, 4, 5, or more of the aforementionedpolymer compounds.

In some embodiments, the anchor A further comprises an ionic chemicalgroup, a material with a hydrophilic moiety, or a material with ahydrophobic moiety, e.g., an aliphatic chain or an aliphatic alcohol. Inembodiments wherein the anchor comprises an ionic chemical group, theionic chemical group may be positively or negatively charged. In someembodiments, the anchor region comprises a reactive moiety for covalentattachment to a support material such as a photoactive phenylazide or anepoxide group. See, U.S. Patent Application Publication No.2016/0159777.

Methods of introducing reactive groups into peptidoglycans and/or otherglycosidic compounds such as polysaccharide, cellulose, glycans, etc.,are known in the art. See US Patent Application Publication No.2005/011261.

Indicators

In some embodiments, the chemical entities comprise one or moreindicators, e.g., at least 1, at least 2, at least 3, at least 4, ormore of indicators. Such compositions may include, for example, aplurality of substrates conjugated to the same gel polymer or differentgel polymers.

In certain embodiments, the indicators are labeled. The term “label,” asused herein, refers to any substance attached to an epitope bindingagent, or other substrate material, in which the substance is detectableby a detection method. Non-limiting examples of suitable labels includeluminescent molecules, chemiluminescent molecules, fluorochromes,fluorescent quenching agents, colored molecules, radioisotopes,scintillants, biotin, avidin, streptavidin, protein A, protein G,antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myctags, heavy metals, and enzymes (including alkaline phosphatase,peroxidase, and luciferase). Methods for attaching the labels to theanchor compounds are described in the Examples.

In certain embodiments, the indicators are labeled with a label which isa detectable label. A detectable label is a moiety, the presence ofwhich can be ascertained directly or indirectly. Generally, detection ofthe label involves the creation of a detectable signal such as forexample an emission of energy. The label may be of a chemical, peptideor nucleic acid nature although it is not so limited. The nature oflabel used will depend on a variety of factors, including the nature ofthe analysis being conducted, the type of the energy source and detectorused and the type of polymer, analyte, probe and primary and secondaryanalyte-specific binding partners.

In a particular embodiment, the label is sterically and chemicallycompatible with the constituents to which it is bound, e.g., the anchorregion. In particular, the label is of the shape and size that it doesnot hinder enzyme recognition site (S) and/or enzyme-reactive region(R).

In another embodiment, the indicator or a motif therein attached to theanchor is a substrate for a lipase, esterase, peroxidase, oxidase,glycosidase, glucuronidase, protease, lactamase, glucosidase,galactosidase, or a combination thereof.

In one embodiment, the indicator or a motif therein attached to theanchor is a substrate for a protease. Particularly, the indicator ormotif therein attached to the anchor is a substrate for elastase.

In another embodiment, the indicator or a motif therein attached to theanchor is a substrate for a protease selected from the group consistingof elastase, cathepsin G, protease 3C or myeloperoxidase (MAO), or acombination thereof.

In another embodiment, the indicator or a motif therein attached to theanchor is a substrate for a glycosidase which is lysozyme and a proteaseselected from the group consisting of elastase, cathepsin G ormyeloperoxidase (MAO), or a combination thereof.

In some embodiments, the enzyme-labile or enzyme-reactive regioninteracts with one or more target enzymes selected from napsin (aspartylprotease), glucosylceramidase glucuronidase, palmitoyl proteinthioesterase, Cathepsins A, B, D, G, L, S, Z; acid ceramidase,lactoferrin (LF), lysozyme, myeloperoxidase (MPO), elastase, cathepsins,and proteinase-3C.

In one embodiment, the indicator (I) or a motif therein attached to theanchor is a peroxidase substrate, an arylamine, an amino phenol, anaminophenyl ether, an indoxyl, a neutral dye, a charged dye, ananoparticle, or a colloidal gold particle.

In some embodiments, the indicator (I) or a motif therein attached tothe anchor is a peroxidase substrate. In some embodiments, theperoxidase substrate is selected from p-aminophenol, ABTS (2,2inophenol,ABTS (strate. In some embodiments, acid) diammonium salt),3,3′-diaminobenzidine, 3,4 diaminobenzoic acid, DCPIP,N,N-dimethyl-p-phenylenediamine, o-dianisidine, p-phenylenediamine,4-chloro-1-naphthol, o-phenylenediamineN-(4-aminobutyl)-N-ethylisoluminol, 3-amino-9-ethylcarbazole,4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), indoxyl, indigo,Fast Blue RR, 4-chloro-7-nitrobenzofurazan. In some embodiments, theindicator (I) or a label attached thereto is an arylamine. In someembodiments, the indicator (I) or a label attached thereto is an aminophenol. In some embodiments, the indicator (I) or a label attachedthereto is an aminophenol ether. In some embodiments, the indicator (I)or a label attached thereto is an indoxyl. In some embodiments, theindicator (I) or a label attached thereto is a neutral dye. In someembodiments, the indicator (I) or a label attached thereto is a chargeddye. In some embodiments, the charged dye is selected from remazolebrilliant blue, toluidine blue, reactive black 5, remazol brilliantblue, reactive violet 5, and reactive orange 16, or a hydrolytic orammonolytic derivatives thereof. In some embodiments, the charged dye isremazole brilliant blue, or a hydrolytic or ammonolytic derivativesthereof. In some embodiments, the charged dye is toluidine blue. In someembodiments, the charged dye is reactive black 5, or a hydrolytic orammonolytic derivatives thereof. In some embodiments, the charged dye isreactive violet 5, or hydrolytic or ammonolytic derivatives thereof. Insome embodiments, the charged dye is reactive orange 16, or hydrolyticor ammonolytic derivatives thereof. In some embodiments, the indicator(I) or a label attached thereto is a dichlorotriazine-based reactive dyesuch as reactive blue 4, reactive red 120, reactive blue 2, reactivegreen 19 and reactive brown 10. In some embodiments, thedichlorotriazine-based reactive dye appears black.

In some embodiments, the indicator (I) or a label attached thereto is areactive dye containing a sulfonylethyl-hydrogensulphate-reactive-group. In some embodiments, the reactivedye is reactive black 5, remazol brilliant blue, reactive violet 5 orreactive orange 16. In some embodiments, the reactive dye is reactiveblack 5. In some embodiments, the reactive dye is remazol brilliantblue. In some embodiments, the reactive dye is reactive violet 5. Insome embodiments, the reactive dye is reactive orange 16. In someembodiments, the reactive dye is reactive black 5, remazol brilliantblue, or reactive violet 5. In some embodiments, the reactive dye isreactive black 5 or remazol brilliant blue.

In some embodiments, the indicator (I) or a label attached thereto is ananoparticle. In some embodiments, the indicator (I) or a label attachedthereto is a colloidal gold particle. In some embodiments, the indicator(I) or a label attached thereto is a charged dye, an indole derivative,or a luminol derivative.

Particularly, the indicator or a motif therein attached to the anchorcomprises a dye containing a sulfonylethyl-hydrogensulphate-reactive-group, e.g., reactive black 5, remazolbrilliant blue, reactive violet 5 or reactive orange 16, or acombination thereof; or a dye containing a dichlortriazinereactive-group, e.g., reactive blue 4, reactive red 120, reactive blue2, reactive green 19 and reactive brown 10, or a combination thereof.

Anchor-Indicator Conjugates

In various enzymes an anchor A is conjugated with the indicator Idirectly, e.g., via an glycosidic linkage. The anchor portion of theconjugate is selected from the group consisting of, e.g., peptidoglycanor chitosan or a polysaccharide and the indicator I is selected from thegroup consisting of a dye containing asulfonylethyl-hydrogensulphate-reactive-group or a dye containing adichlortriazine reactive-group.

Markers

Embodiments described herein may utilize chemical moieties that assayfor various biological markers present in a chronic or infected wound.In one embodiment, the marker is a wound-specific marker, which is anenzyme selected from the group consisting of hydrolases, proteases,esterases, and peroxidases.

As used herein, a “wound specific enzyme” is an enzyme that isdifferentially expressed in a wound. By “differential expression” it ismeant that the level or the activity of the enzyme is higher or lower inthe wound microenvironment compared to other sites, e.g., normal tissueor surrounding tissue. Particularly, differential expression implieshigher level of expression or activity of the enzyme in the woundmicroenvironment compared to normal or unwounded tissue. Differentialexpression of enzyme may be analyzed by routine means. For example,levels of enzyme in a sample may be analyzed by ELISA assays or otherimmunoassays. Activities of the enzyme may be analyzed by measuringrates of loss of a substrate and/or rates of formation of the product,e.g., using mass spectroscopy or HPLC. Such techniques are known in theart and are described in the Examples section.

In one embodiment, the marker is a hydrolase. As used herein, a“hydrolase” or “hydrolytic enzyme” is an enzyme that catalyzes thehydrolysis of a chemical bond, e.g., esterases and nucleases (breakester bonds); glycolases (break glycosidic linkers); peptidases (breakpeptide bonds), etc.

In one specific embodiment, the wound-specific glycoside hydrolase islysozyme. Lysozyme (UNIPROT accession Nos. P61626 [human] and P08905[mouse]) is a glycoside hydrolase and its main function is to destroythe cell walls of bacteria. It hydrolyses the (1→4)-β-linkages betweenN-acetylmuramic acid and N-acetyl-D-glucosamine residues inpeptidoglycan and also between N-acetyl-D glucosamine residues inchitodextrin. The natural substrate for lysozyme is the peptidoglycanlayer of bacterial cell walls. However, a variety of low molecular masssubstrates including murein degradation products as well as syntheticcompounds have been used for various photometric, isotopic, andimmunological lysozyme assays. Holtje et al., EXS, 75:105-10,1996. Seealso Sigma Catalog Number M5639 and Sigma Catalog Number N8638.

In one embodiment, the individual components of the chemical moiety havebeen adapted for recognition by wound-specific hydrolase, e.g., awound-specific lysozyme.

Alternately or additionally, the individual components of the chemicalmoiety can be modified for recognition by other wound specific enzymes.In one embodiment, the additional wound specific enzyme is a protease.As used herein, a “wound specific protease” is a protease that isdifferentially expressed in a wound. By “differential expression” it ismeant that the level or the activity of the protease is higher or lowerin the wound microenvironment compared to other sites, e.g., normaltissue or surrounding tissue. Particularly, differential expressionimplies higher level of expression or activity of the protease in thewound microenvironment compared to unwounded tissue. Differentialexpression of proteases may be analyzed by routine means. For example,levels of proteases in a sample may be analyzed by ELISA assays or otherimmunoassays. Activities of the proteases may be analyzed by measuringrates of loss of a peptide substrate and/or rates of formation of theproduct, e.g., using mass spectroscopy or HPLC. Such techniques areknown in the art and are described in the Examples section.

In one embodiment, the wound-specific protease is cathepsin G (UNIPROTaccession Nos. P08311 [human] and P28293 [mouse]), which is one of thethree serine proteases of the chymotrypsin family that are stored in theazurophil granules. Cathepsin G-specific substrates have the sequenceAla-Ala-Pro-Phe [SEQ ID NO: 3] or Ala-Ala-Pro-Met [SEQ ID NO: 4] (SigmaAldrich Catalog Nos. S7388 and M7771).

In another embodiment, the wound specific protease is elastase (e.g.,human neutrophil elastase or HNE) (UNIPROT accession Nos. P08246 [human]and Q3UP87 [mouse]). HNE is a serine proteinase in the same family aschymotrypsin and has broad substrate specificity. Secreted byneutrophils and macrophages during inflammation, it destroys bacteriaand host tissue. In one embodiment, the substrate for detecting HNE hasa core sequence Alanine-Alanine-Proline-Valine (AAPV) [SEQ ID NO: 5]. Inanother embodiment, the substrate for HNE is Ala-Pro-Glu-Glu-Ile [SEQ IDNO: 6]/Met-Arg-Arg-Gln [SEQ ID NO: 7] (APEEI/MRRQ) (Kasperkiewicz etal., PNAS USA, 111(7): 2518-2523, 2014; Korkmaz et al., Methods MolBiol, 844: 125-138, 2012).

In one embodiment, the enzyme-labile region comprises a peptide that isliable to elastase. Under this embodiment, the chromogenic indicator forelastase would be high contrast and thus serve as a clear indicator whenused in situ in medicinal products.

The ideal substrate would make a blue, violet or deep green color. Itwould also be fixed in a sterically permissible position with highturnover. The state of the art is the opposite. Available substratescontain a p-nitrophenol group, which is low molecular weight but givesrise to a yellow soluble chromophore. Most skilled investigators regardthat the substrate should be soluble in water, reasoning that this isthe most likely way that the substrate will find its way to the activesite.

In contrast the embodiments described herein depart from that generalrationale. It was contemplated that elastase digests a solid phasesubstrate, namely structural proteins, which are, by definition, notsoluble, that a substrate specific to it would have to be adaptedaccordingly. As such, both the color of the indicator and the systemsthat they could be employed with, e.g., electronically detection, wereadapted to the wound environment.

Therefore, contrary to the art teachings to employ soluble substrates,embodiments described herein contemplate use of a low water soluble,elastase substrates that give rise to Blue, violet or Green colors.

Still in a further embodiment, the wound-specific enzyme is peroxidase,more specifically, a myeloperoxidase (MPO). MPO (UNIPROT accession Nos.P05164 [human] and PI 1247 [mouse]) is a peroxidase found in neutrophilgranulocytes. In the presence of hydrogen peroxide (H202) and a halide(most commonly chloride) it produces the antimicrobial substanceshypochlorite, singlet oxygen (102), chlorine (C12) and hydroxyl radicals(OH·). MPO can be detected using tetramethylbenzidine or4-Benzoylamino-2,5-dimethoxyaniline. See, Andrews et al., Anal Biochem,127(2):346-50, 1982; Klebanoff et al., J. Leukocyte Biol, 77, 598-625,2005.

Still in a further embodiment, the wound-specific enzyme is a bacterialenzyme, more specifically, beta-lactamases (β-lactamases). B-lactamases(AccessionGO:0008800) are hydrolase enzymes (EC 3.5.2.6) produced bybacteria (also known as penicillinase) that provide multi-resistance toβ-lactam antibiotics such as penicillins, cephamycins, and carbapenems(ertapenem). Through hydrolysis, the lactamase enzyme breaks theβ-lactam ring open.

Still in a further embodiment, the wound-specific enzyme is a viralenzyme, more specifically, protease 3C. These proteases are encoded byenteroviruses, rhinoviruses, aphtoviruses and cardioviruses, whichgenera all cause a wide range of infections for humans and othermammals. Accordingly, protease 3C can be employed as a marker for woundinfection.

Still in a further embodiment, the wound-specific enzyme is MPO (UNIPROTaccession Nos. P05164 [human] and PI 1247 [mouse]) is a peroxidase foundin neutrophil granulocytes. In the presence of hydrogen peroxide (H202)and a halide (most commonly chloride) it produces the antimicrobialsubstances hypochlorite, singlet oxygen (102), chlorine (C12) andhydroxyl radicals (OH·). MPO can be detected using tetramethylbenzidineor 4-Benzoylamino-2,5-dimethoxyaniline. See, Andrews et al., AnalBiochem, 127(2):346-50, 1982; Klebanoff et al., J. Leukocyte Biol, 77,598-625, 2005

Enzyme Recognition Site (R)

Insofar as embodiments disclosed herein relate to the specific detectionof wound-specific markers, disclosed herein are substrates containingenzyme recognition sites (R) for the wound-specific markers. Thus, inone embodiment, the chemical moiety comprises an anchor region A or anindicator (I) comprising a recognition site for a wound-specific enzyme,e.g., an enzyme cleavage site.

In one embodiment, the enzyme recognition site comprises glycosidicbonds. As used herein, a “glycosidic bond” is formed between thehemiacetal or hemiketal group of a saccharide (or a molecule derivedfrom a saccharide) and the hydroxyl group of some compound such as analcohol. A substance containing a glycosidic bond is a glycoside. Theterm “glycoside” is now extended to also cover compounds with bondsformed between hemiacetal (or hemiketal) groups of sugars and severalchemical groups other than hydroxyls, such as —SR (thioglycosides), —SeR(selenoglycosides), —R1R2 (N-glycosides), or even —CR1R2R3(C-glycosides).

In one embodiment, the chemical moieties disclosed herein contain one ormore glycosidic bonds which are cleaved by glycolases. In one specificembodiment, the chemical moieties comprise a glycosidic bond linkinganchor A and the indicator I, either directly or via another group.Particularly, the anchor A and the indicator I are directly linked viaone or more glycosidic bonds, in which case, the chemical entity iscleaved by the glycolase and therefore can be used in detecting theglycolase.

In one embodiment, the indicator molecule comprises anenzymatically-cleavable peptide comprising a peptide bond. As usedherein, a “peptide bond” is formed by the condensation reaction betweentwo amino acids, wherein the acid moiety of one reacts with the aminomoiety of the other to produce a peptide bond (—CO—NH—) between the twoamino acids. The individual peptides provide a motif for the recognitionby a sequence-specific protease. As used herein, the term“sequence-specific protease” means a protease recognizing a specificsequence of a peptide for its digesting (for example, caspase), and isdistinguished from a generic protease (for example, trypsin) thatsequentially decomposes a peptide from one end thereof or digest apeptide in a sequence-nonspecific manner. For sequence specificity, theamino acid sequence of the peptide substrate may comprise four or moreamino acid (a.a.) residues.

As used herein, the term “peptide” includes a natural peptide comprisinga linear chain or branched amino acids, peptidomimetics, as well aspharmaceutically acceptable salts thereof. Typically, a peptidecomprises a plurality of amino acid residues, e.g., 2, 3, 4, 5, 6, 8,10, or more amino acid residues which are bonded to each other viacovalent bonds, e.g., a peptide bond. “Amino acid residue” means theindividual amino acid units incorporated into the peptides of thedisclosure. As used herein, the term “amino acid” means a naturallyoccurring or synthetic amino acid, as well as amino acid analogs,stereoisomers, and amino acid mimetics that function similarly to thenaturally occurring amino acids. Included by this definition are naturalamino acids such as: (1) histidine (His) (2) isoleucine (He) (3) leucine(Leu) (4) lysine (Lys) (5) methionine (Met) (6) phenylalanine (Phe) (7)threonine (Thr) (8) tryptophan (Trp) (9) valine (Val) (10) arginine(Arg) (11) cysteine (Cys) (12) glutamine (Gin) (13) glycine (Gly) (14)proline (Pro) (15) serine (Ser) (16) tyrosine (Tyr) (17) alanine (Ala)(18) asparagine (Asn) (19) aspartic acid (Asp) (20) glutamic acid (Glu)(21) selenocysteine (Sec); including unnatural amino acids: (a)citrulline; (b) cystine; (c) gama-amino butyric acid (GAB A); (d)omithine; (f) theanine and amino acid derivatives such as betaine;carnitine; carnosine creatine; hydroxytryptophan; hydroxyproline;N-acetyl cysteine; S-Adenosyl methionine (SAM-e); taurine; tyramine.Among these, amino acids containing reactive side chains, e.g.,cysteine, serine, threonine, lysine, arginine, aspartate/asparagine,glutamate/glutamine, glycine, alanine, etc. are particularly employedfor modification of the substrate.

In some embodiments, the chemical entities contain one or moreenzyme-labile or enzyme-reactive regions (R) for the detection ofwound-specific enzymes.

In one embodiment, wherein the enzyme is a glycosidase such as lysozyme,the enzyme-labile or enzyme-reactive region comprises an acyl chitosanof at least 3 glucosamine or N-acetylglucosamine or peptidoglycan units,which are optionally acetylated. The enzyme reactive site may contain,for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or more units ofglucosamine or N-acetyl glucosamine or peptidoglycan units. In oneembodiment, the R comprises at least 3 glucosamine orN-acetylglucosamine or a combination thereof, wherein the glucosamineand/or N-acetyl glucosamine are optionally acetylated. In anotherembodiment, the enzyme-labile or enzyme-reactive region comprisespeptidoglycan, wherein the peptidoglycan is optionally acetylated.

In some embodiments, the chemical moieties comprise enzyme reactivesites (R) for one or more wound-specific protease disclosed above, e.g.,cathepsin G, and myeloperoxidase, elastase or a combination thereof. Asused herein, the term “reactive site for a protease” means a peptidecomprising an amino acid sequence of a protein, which is recognized bythe protease as a substrate for its protease activity, e.g., as asubstrate that can be cleaved into one or more products. In someembodiments, the chemical entities comprise a peptide region comprisinga peptide sequence comprising a plurality of amino acids. The term“plurality” means two or more units, e.g., amino acids, although theindividual units need not be structurally and/or functionally different.Typically, the indicator region (I) of the chemical entity comprises thepeptide which serves as the enzyme reactive site for the wound-specificprotease.

In one embodiment, the enzyme-labile or enzyme-reactive region comprisesa peptide that is labile to elastase, cathepsin G, myeloperoxidase or acombination thereof.

In one embodiment, the enzyme-labile region comprises a peptide that isliable to elastase. Under this embodiment, the chromogenic indicator forelastase would be high contrast and thus serve as a clear indicator whenused in situ in medicinal products.

The ideal substrate would make a blue, violet or deep green color. Itwould also be fixed in a sterically permissible position with highturnover. The state of the art is the opposite. Available substratescontain a p-nitrophenol group, which is low molecular weight but givesrise to a yellow soluble chromophore. Most skilled investigators regardthat the substrate should be soluble in water, reasoning that this isthe most likely way that the substrate will find its way to the activesite.

In contrast the embodiments described herein depart from that generalrationale. It was contemplated that elastase digests a solid phasesubstrate, namely structural proteins, which are, by definition, notsoluble, that a substrate specific to it would have to be adaptedaccordingly. As such, both the color of the indicator and the systemsthat they could be employed with, e.g., electronically detection, wereadapted to the wound environment.

Therefore, contrary to the art teachings to employ soluble substrates,embodiments described herein contemplate use of a low water soluble,elastase substrates that give rise to Blue, violet or Green colors.

The peptide sequence is often considered to be important for proteasesubstrates, however, Elastase has a very general hydrolytic potentialand accepts very many substrates. This is because it is also involved inanti-microbial defense and immune cell translocation in many tissues. Inthis regard, it can cut many different peptides. It Cuts well between A,F, V or M, and a simple amino acid with limited side chain complexity.AAPV [SEQ ID NO: 5], AAPF [SEQ ID NO: 3], AAAA [SEQ ID NO: 8] are allexamples of well recognized targets. What is apparent is that withincreasing distance on the N-terminus, the amino acids play a lessimportant role. On the C-terminus of the cut site, there is no obviousconsensus, however, less complexity appears to be preferred.

More important in this case are two factors:

-   -   Distance from the anchor    -   Nature of the chromophore

If the Anchor site is too close, the action of the enzyme is inhibited.Therefore, ideally, there are 4 or more aminoacids between the cut siteand the anchor.

The chromophore, and notably its charge are important. Neutralchromophores are preferred to positively charged moieties, and these arepreferred vs. negatively charged chromophores or dyes. A high density ofnegative charge near the cut site inhibits the enzyme. Thus, wherenegative dyes are used, they are preferably spaced 2 or more aminoacidsC-terminal form the cut site.

Longer amino acid sequences are generally less hindered but they arealso less economical

In one embodiment, the enzyme-labile or enzyme-reactive region comprisesa peptide comprising an amino acid sequence of:

X_(y)AAPX_(y)-Z,

-   -   wherein each X is independently any amino acid,    -   y is each, independently, an integer between 0 and 200, and    -   Z comprises a detectable label.

In one embodiment, the enzyme-labile or enzyme-reactive region comprisesa peptide comprising an amino acid sequence of:

X_(y)AAPX_(y)-L-Z,

-   -   wherein each X is independently any amino acid,    -   y is each, independently, an integer between 0 and 200, and    -   Z comprises a detectable label.

In another embodiment, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence of:

X_(y)AAP(V/F/A)X_(y)-Z,

-   -   wherein each X is independently any amino acid,    -   y is each, independently, an integer between 0 and 200, and    -   Z comprises a detectable label.

In yet another embodiment, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence of:

X_(y)AAP(V/F/A)X_(y)-L-Z,

-   -   wherein each X is independently any amino acid,    -   y is each, independently, an integer between 0 and 200,    -   L is a linking moiety, and    -   Z comprises a detectable label.

In another specific embodiment, the reactive region R comprises thepeptide sequence X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z, wherein X, L and Zare each, as described above, and y is, each, independently an integerfrom 1 to 50.

Still in a further embodiment, the reactive region R comprises thepeptide sequence X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z, wherein X, L and Zare each, as described above, and y is, each, independently an integerfrom 1 to 10.

Particularly, the reactive region R comprises the peptide sequenceX_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z, X_(y)-AAP(V/F/A)X_(y)-Z orX_(y)AAP(V/F/A)X_(y)-L-Z, wherein X, L and Z are each, as describedabove, and y is, each, independently an integer from 1 to 6.

In one embodiment, each of the aforementioned peptides comprising thesequence X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z, X_(y)AAP(V/F/A)X_(y)-Z orX_(y)AAP(V/F/A)X_(y)-L-Z, are each, individually, labile to elastase.

In some embodiments, one or more of the amino acids in the amino acidsequence X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z, X_(y)AAP(V/F/A)X_(y)-Z orX_(y)AAP(V/F/A)X_(y)-L-Z is protected, e.g., with an amine protectiongroup, for example, fluorenylmethyloxycarbonyl (Fmoc).

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide that is labile to cathepsin G.

In one embodiment, the enzyme-labile or enzyme-reactive region comprisesa peptide comprising an amino acid sequence of:

X_(y)N⁴N³N²N¹X_(y)Z, wherein

-   -   X is each, independently, any amino acid;    -   y is each independently a number selected from 0 to 6;    -   N⁴ is selected from alanine, glycine, valine, and glutamine;    -   N³ is selected from alanine, glycine, proline, lysine, and        serine;    -   N² is selected from proline, alanine, and glycine;    -   N¹ is selected from serine, lysine, phenylalanine, arginine,        leucine, and methionine; and    -   Z comprises a detectable label; and the peptide is labile to        cathepsin G.

In some embodiments, one or more of the amino acids in the amino acidsequence is protected. In some embodiments, one or more of the aminoacids in the amino acid sequence is protected with a t-boc group. Insome embodiments, one of the amino acid in the amino acid sequence isprotected with an fmoc group.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence of

X_(y)N⁴N³N²N¹X_(y)-L-Z, wherein

-   -   X is each, independently any amino acid;    -   y is each, independently, a number selected from 0 to 6;    -   N⁴ is selected from alanine, glycine, valine, and glutamine;    -   N³ is selected from alanine, glycine, proline, lysine, and        serine;    -   N² is selected from proline, alanine, and glycine;    -   N¹ is selected from serine, lysine, phenylalanine, arginine,        leucine, and methionine; and    -   L is a linking moiety; and    -   Z comprises a detectable label.

In one embodiment, each of the aforementioned peptides comprising thesequence X_(y)N⁴N³N²N¹X_(y)-Z and X_(y)N⁴N³N²N¹X_(y)-L-Z, are each,individually, labile to cathepsin G.

In some embodiments, one or more of the amino acids in the amino acidsequence X_(y)N⁴N³N²N¹X_(y)-Z and X_(y)N⁴N³N²N¹X_(y)-L-Z is protected,e.g., with an amine protection group, for example,fluorenylmethyloxycarbonyl (Fmoc).

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peptide comprising an amino acid sequence of (a)X_(y)UUUU_(y)-Z, wherein X is, each, independently any amino acid; y is,each, independently, a number selected from 1 to 50; U is an amino acidselected from LEVLFQ, and Z is a label. Particularly under thisembodiment, y is a number selected from 1 to 10.

In some embodiments, one or more of the amino acids in the amino acidsequence (a) XyUUUUy-Z is protected, e.g., with a t-boc group or an fmocgroup.

In some embodiments, the amino acid sequence X_(y)UUUU_(y)-Z is liableto a viral 3C protease.

Detectable Label Z

In some embodiments, Z is a peroxidase substrate, an arylamine, an aminophenol, an aminophenyl ether, an indoxyl, a neutral dye, a charged dye,a nanoparticle, or a colloidal gold particle.

In some embodiments, Z is a peroxidase substrate selected fromp-aminophenol, ABTS (2,2inophenol, ABTS (s, the peroxidase substrateacid) diammonium salt), 3,3′-diaminobenzidine, 3,4 diaminobenzoic acid,DCPIP, N,N-dimethyl-p-phenylenediamine, o-dianisidine,p-phenylenediamine, 4-chloro-1-naphthol, o-phenylenediamineN-(4-aminobutyl)-N-ethylisoluminol, 3-amino-9-ethylcarbazole,4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), indoxyl, indigo,Fast Blue RR, 4-chloro-7-nitrobenzofurazan.

In some embodiments, Z is an arylamine, an amino phenol, an aminophenolether, an indoxyl, a neutral dye, a charged dye selected from remazolebrilliant blue, toluidine blue, reactive black 5, remazol brilliantblue, reactive violet 5, and reactive orange 16, or a hydrolytic orammonolytic derivatives thereof. Particularly, Z is a charged dyeselected from remazole brilliant blue; toluidine blue; reactive black 5or a hydrolytic or an ammonolytic derivative thereof.

In some embodiments, Z is a dichlorotriazine-based reactive dye such asreactive blue 4, reactive red 120, reactive blue 2, reactive green 19and reactive brown 10. In some embodiments, the dichlorotriazine-basedreactive dye appears black. In some embodiments, Z is a reactive dyecontaining a sulfonyl ethyl-hydrogensulphate-reactive-group.

In some embodiments, Z is a nanoparticle. In some embodiments, Z is acolloidal gold particle.

In some embodiments, Z is a charged dye, an indole derivative, or aluminol derivative.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a phenol, an amino phenol, an aminophenyl ether, an indoxyl,or a quinone. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises a phenol. In some embodiments, the enzyme-labile orenzyme-reactive region comprises an amino phenol. In some embodiments,the enzyme-labile or enzyme-reactive region comprises an amino phenolether. In some embodiments, the enzyme-label or enzyme-reactive regioncomprises an indoxyl. In some embodiments, the enzyme-labile orenzyme-reactive region comprises a quinone. In some embodiments, theenzyme-labile or enzyme-reactive region reacts with myeloperoxidase butdoes not react with heme.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a peroxidase substrate, an arylamine, an amino phenol, aneutral dye, a charged dye, a nanoparticle, or a colloidal goldparticle. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises a peroxidase substrate. In some embodiments, theperoxidase substrate is selected from p-aminophenol, ABTS(2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt),3,3′-diaminobenzidine, 3,4 diaminobenzoic acid, DCPIP,N,N-dimethyl-p-phenylenediamine, o-dianisidine, p-phenylenediamine,4-chloro-1-naphthol, o-phenylenediamineN-(4-aminobutyl)-N-ethylisoluminol, 3-amino-9-ethylcarbazole,4-aminophthalhydrazide, 5-aminosalicylic acid,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and4-chloro-7-nitrobenzofurazan, Fast Blue RR, N-(2-hydroxy)tetradecyl-FastBlue RR. In some embodiments, the enzyme-labile or enzyme-reactiveregion comprises an arylamine. In some embodiments, the enzyme-labile orenzyme-reactive region comprises an amino phenol. In some embodiments,the enzyme-labile or enzyme-reactive region comprises a neutral dye. Insome embodiments, the enzyme-labile or enzyme-reactive region comprisesa charged dye. In some embodiments, the charged dye is selected fromremazole brilliant blue, toluidine blue, reactive black 5, remazolbrilliant blue, reactive violet 5, and reactive orange 16, or hydrolyticor ammonolytic derivatives of each of these. In some embodiments, thecharged dye is remazole brilliant blue, or hydrolytic or ammonolyticderivatives thereof. In some embodiments, the charged dye is toluidineblue. In some embodiments, the charged dye is reactive black 5, orhydrolytic or ammonolytic derivatives thereof. In some embodiments, thecharged dye is reactive violet 5, or hydrolytic or ammonolyticderivatives thereof. In some embodiments, the charged dye is reactiveorange 16, or hydrolytic or ammonolytic derivatives thereof.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a dichlorotriazine-based reactive dye such as reactive blue 4,reactive red 120, reactive blue 2, reactive green 19 and reactive brown10.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a nanoparticle. In some embodiments, Z is a colloidal goldparticle.

In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises a charged dye, an indole derivative, or a luminol derivative.In some embodiments, the enzyme-labile or enzyme-reactive regioncomprises an indole derivative. In some embodiments, the enzyme-labileor enzyme-reactive region comprises a luminol derivative.

In some embodiments, the indicator region comprises a dye that presentsa visible color change in normal ambient lighting. In some embodiments,the dye has a contrasting color to wound products, which are commonlyred, yellow, or brown. In further embodiments, the dye is violet, blueor dark green. In some embodiments, the dye is violet. In someembodiments, the dye is blue. In some embodiments, the dye is darkgreen. In some embodiments, the dye has low molecular weight, ischarged, contains reactive or linkable groups, is stable to gammairradiation, and is deeply colored. In some embodiments, the dye isselected from cibracron series dyes, azo dyes, and remazol dyes, orhydrolytic or ammonolytic derivatives thereof. In some embodiments, thedye is selected from cibracron series dyes. In some embodiments, the dyeis selected from azo dyes. In some embodiments, the dye is selected fromremazol dyes, or hydrolytic or ammonolytic derivatives thereof. In someembodiments, the dye is selected from rhodamine, coumarin, cyanine,xanthene, polymethine, pyrene, dipyrromethene borondifluoride,napthalimide, a phycobiliprotein, peridinium chlorophyll proteins,fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594,tetramethylrhodamine, a carboxyrhodamine, carboxyrtiodamine 6F,carboxyrtiodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633,DyLight 649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP(peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6-)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350,Alex Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-aceticacid, BODIPY® FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568,BODIPY® 630/650, BODIPY® 650/665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR,and dimethylaminoazobenzenesulfonic acid (dabsyl), or conjugatesthereof, or combinations thereof.

In some embodiments, the indicator region comprises adichlorotriazine-based reactive dye such as reactive blue 4, reactivered 120, reactive blue 2, reactive green 19 and reactive brown 10. Insome embodiments, the dichlorotriazine-based reactive dye appears black.

In some embodiments, the indicator region comprises the reaction productof a reactive dye containing asulfonylethyl-hydrogensulphate-reactive-group. In some embodiments, thereactive dye is reactive black 5, remazol brilliant blue, reactiveviolet 5 or reactive orange 16. In some embodiments, the reactive dye isreactive black 5. In some embodiments, the reactive dye is remazolbrilliant blue. In some embodiments, the reactive dye is reactive violet5. In some embodiments, the reactive dye is reactive orange 16. In someembodiments, the reactive dye is reactive black 5, remazol brilliantblue, or reactive violet 5. In some embodiments, the reactive dye isreactive black 5 or remazol brilliant blue.

In some embodiments, the indicator region comprises a particle (e.g.,colloidal metal or quantum dots) that present color changes in normalambient lighting. In some embodiments, the indicator region comprises ananoparticle. In some embodiments, the indicator region comprises acolloidal gold particle.

In some embodiments, the indicator region comprises a dye that presentsa visible color change under UV light. In some embodiments, theindicator region comprises a dye that is fluorescent. In someembodiments, the indicator region comprises a dye that is luminescent.

In some embodiments, the indicator region comprises an enzyme-reactivemoiety. In some embodiments, the enzyme-reactive moiety interacts withan accessory enzyme to produce a product that is visible to the nakedeye or detectable by electronic means. In some embodiments, theenzyme-reactive moiety interacts with an accessory enzyme to produce aproduct that is visible to the naked eye. In some embodiments, theenzyme-reactive moiety interacts with an accessory enzyme to produce aproduct that is detectable by electronic means. In some embodiments, theindicator region comprises an indoxyl glycoside that is cleaved byhexaminidase, glucuronidase, glucosidase or galactosidase depending onthe terminal sugar used, to produce indigo. In some embodiments, theindicator region comprises a phenol that is oxidized by an accessoryenzyme to produce a visible product. In some embodiments, the indicatorregion comprises a phenol that is oxidized by laccase to produce avisible product. In some embodiments, the indicator region comprises ametallo motif that is detectable by electronic means. In someembodiments, the indicator region comprises a ferrocene or ferroceneanalog that is detectable by electronic means. In some embodiments, theaccessory enzyme is selected from lipase, esterase, hexosaminidase,peroxidase, oxidase, glycosidase, glucosidase, and laccase. In someembodiments, the accessory enzyme is not present in the wound fluid. Insome embodiments, the accessory enzyme is present in the wound fluid. Insome embodiments, the enzyme-reactive moiety interacts with an accessoryenzyme to produce a product that is visible under UV light.

Chemical Entities Containing a Plurality of Enzyme Recognition Sites (R)

In further embodiments, disclosed herein are chemical entitiescontaining the anchor A, the indicator I, which individually or togethercomprise a plurality of enzyme recognition sites (R). Typically, suchchemical entities are employed to assay for a plurality of enzymes,e.g., a combination comprising at least one protease and at least oneglycosidase.

In one embodiment, disclosed herein are chemical entities containing theanchor A, the indicator I, which individually or together comprise aplurality of enzyme recognition sites (R), wherein at least one reactivesite is specific for a glycosidase, e.g., lysozyme; and at least oneenzyme reaction site is specific for a protease selected from the groupconsisting of lipase, esterase, peroxidase, oxidase, glycosidase,glucuronidase, glucosidase, galactosidase, Napsin (aspartyl protease),Glucosylceramidase glucuronidase, palmitoyl protein thioesterase,cathepsins A, B, D, G, L, S, Z; acid ceramidase, lactoferrin (LF),lysozyme, myeloperoxidase (MPO), elastase, lactamase, cathepsins, andproteinase-3C or a combination thereof. The individual reaction sitesand recognition sites for these enzymes have been described previously.

In one embodiment, disclosed herein are chemical entities containing theanchor A, the indicator I, which individually or together comprise aplurality of enzyme recognition sites (R), wherein at least one reactivesite is specific for a protease elastase; and at least one enzymereaction site is specific for a protease selected from the groupconsisting of myeloperoxidase (MPO), cathepsins, and proteinase-3C. Theindividual reaction sites and recognition sites for these enzymes havebeen described previously.

In one embodiment, disclosed herein are chemical entities containing theanchor A, the indicator I, which individually or together comprise aplurality of enzyme recognition sites (R), wherein at least one reactivesite is specific for a protease elastase; and at least one enzymereaction site is specific for a microbial enzyme selected from the groupconsisting of beta-lactamase and proteinase-3C. The individual reactionsites and recognition sites for these enzymes have been describedpreviously

In one embodiment, disclosed herein are chemical entities containing theanchor A, the indicator I, which individually or together comprise aplurality of enzyme recognition sites (R), wherein at least one reactivesite is specific for an elastase; and at least one enzyme reaction siteis specific for a protease selected from the group consisting of MPO.The individual reaction sites and recognition sites for these enzymeshave been described previously.

In one embodiment, disclosed herein are chemical entities containing theanchor A, the indicator I, which individually or together comprise aplurality of enzyme recognition sites (S) and enzyme reaction sites (R),wherein at least one reactive site is specific for a protease elastase;and at least one enzyme reaction site is specific for a proteaseselected from the group consisting of proteinase 3C. The individualreaction sites and recognition sites for these enzymes have beendescribed previously.

Owing to the greater predictive power of employing a combination ofenzyme substrates, it is contemplated that the diagnostic utility ofchemical entities comprising a plurality of reaction and recognitionsites, as outlined above, will be greatly enhanced compared to entitiescomprising unitary (e.g., single type) of reaction and recognitionsites. At the very least, entities comprising a plurality ofreaction/recognition sites will permit diagnosis of at least 2, at least3, at least 4 or more markers simultaneously. By the way of example,host elastase and/or MPO activity at the wound situs may be detected andmonitored simultaneously with pathogen-derived markers (e.g.,beta-lactamase or viral protease 3C-specific reaction sites) using themultiplex chemical entities disclosed herein.

Support Material

In some embodiments, the anchor region (A) of the chemical entity bindsthe chemical entity to a support material, e.g., via covalentinteraction, ionic interaction, hydrophobic interaction, electrostaticinteractions, hydrogen bonding interactions, physiochemicalinteractions, van der Waal forces, Lewis-acid/Lewis-base interactions,or combinations thereof.

In some embodiments, the support matrix comprises dextran, agarose,silica, synthetic polymer, or dextran, agarose, silica, or syntheticpolymer covalently coupled to an antibody, ligand, or epitope tag.

In some embodiments, the anchor region is a polystyrene bead, silica gelbead, polysaccharide bead, polyacrylamide bead, cellulose bead,polysaccharide, derivatized cellulose, polyacrylate, polyethyleneimine,polyacrylamide, UV-activatable reactive group, peptidoglycan, orchitosan derivative, or a combination thereof. In some embodiments, theanchor region binds to a support material after a short period of UVirradiation.

In some embodiments, the chemical entity is printed on or in a supportmaterial such as filter paper or a woven or non-woven material that iscapable of being wet by a wound fluid and which displays capillaryaction. In some embodiments, the reporting entity or chemical entity ischemically bonded onto or into a support material such as filter paperor a woven or non-woven material that is capable of being wet by a woundfluid and which displays capillary action that is similar in alldimensions. In some embodiments, the chemical entity is ionically boundonto or into a support material such as filter paper or a woven ornon-woven material that is capable of being wet by a wound fluid andwhich displays capillary action. In some embodiments, the chemicalentity is covalently bound onto or into a support material such asfilter paper or a woven or non-woven material that is capable of beingwet by a wound fluid and which displays capillary action. Supportmaterial includes, but is not limited to, cellulose, polyamide,polyester, polyacrylate and other similar polymers that are useful asfibers. In some embodiments, the support material is cellulose. In someembodiments, the support material is polyamide. In some embodiments, thesupport material is polyester. In some embodiments, the support materialis polyacrylate.

Additional Moieties

In some instances, the pH of a wound can influence many factors of woundhealing, such as angiogenesis, protease activity, oxygen release, andbacterial toxicity. Chronic nonhealing wounds may have an elevatedalkaline environment. As the wound progresses towards healing, the pH ofthe wound moves to neutral and then becomes acidic. Monitoring of the pHof the wound may provide a method to assess the condition of the wound(e.g., infection or no infection) and aid in determining a wound'sresponse to treatment.

Accordingly, in some embodiments, the chemical entity for the detectionof infection in a wound comprises an indicator region comprising apH-sensitive moiety that presents a visible color change. In oneembodiment, the pH-sensitive moiety presents a visible color change atalkaline pH, e.g., a pH=7.2-9.5; pH=7.2-9.0; pH=7.2-8.5; pH=7.2-8.0;pH=7.5-8.5; pH=7.5-9.0; pH=8.0-9.0. In other embodiments, thepH-sensitive moiety presents a visible color change at pH=7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5, or 0.1 increments thereof.

In some embodiments, the pH-sensitive moiety presents a visible colorchange at neutral pH range, e.g., at pH=6.9, 7.0, or 7.1, or 0.05increments thereof.

In some embodiments, the pH-sensitive moiety presents a visible colorchange at acidic pH, e.g., pH=4.5-6.8; pH=4.5-6.5; pH=5.0-6.8;pH=5.4-6.8; pH=5.4-6.5. In other embodiments, the pH-sensitive moietypresents a visible color change at pH=4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, or 6.9, or 0.1 increments thereof.

In some embodiments, the pH-sensitive moiety is selected from the groupconsisting of bromothymol blue, phenol red, bromophenol red,chlorophenol red, thymol blue, bromocresol green, bromocresol purple;nitrazine yellow; and sulfophthalein dyes or a combination thereof.

Compositions:

Embodiments described herein further relate to compositions containingthe compounds of Formula I. Such compositions may be prepared usingconventional methods.

Once formulated, the resulting stock composition of compounds of FormulaI may be further modified into desired form, e.g., gels, balms, lotions,cream, paste, ointments, etc. using conventional methods, e.g., usingcarriers, gelling agents, emollients, surfactants, humectants, viscosityenhancers, emulsifiers, etc. See, e.g., WO 2013/004953.

Carriers for use in the composition may include, but are not limited to,water, glycerin, diglycerin, glycerin derivatives, glycols, glycolderivatives, sugars, ethoxylated and/or propoxylated esters and ethers,urea, sodium PCA, alcohols, ethanol, isopropyl alcohol, and combinationsthereof. In one embodiment, the carrier is propylene glycol. Typically,the composition contains a carrier in an amount from about 1% by weightof the composition to about 99.9% by weight of the composition, moretypically from about 2% by weight of the composition to about 95% byweight of the composition, and more typically from about 5% by weight ofthe composition to about 90% by weight of the composition.

Thermo-reversible gelling agents are defined as ingredients that aresoluble, partially soluble, or miscible in a hydrophilic carrier atelevated temperatures, such as 50° C., wherein the agents have theability to thicken the carrier when cooled to 25° C., but will be lessviscous at 50° C. when application to a substrate is necessary. Suitablehydrophilic carriers include water, glycols, e.g., propylene glycol.Thermo-reversible gelling agents for use in the composition may includesalts of fatty acids such as sodium stearate, sodium palmitate,potassium stearate. These salts can be added to the composition or canbe created in-situ by addition of the fatty acid and neutralizing withappropriate base. An example of in-situ formation of the composition isto provide stearic acid and sodium hydroxide to produce sodium stearate.Other common hermos-reversible gelling agents could include, e.g.,polyethylene glycols and derivatives such as PEG-20, PEG-150 distearate,PEG-150 pentaerythrityl tetrastearate, disteareth-75 IPDI,disteareth-100 IPDI, fatty alcohols, e.g., cetyl alcohol, fatty acidssuch as stearic acid, hydroxystearic acid and its derivatives, andcombinations thereof.

In addition to the carrier and hermos-reversible gelling agent, thecomposition can contain various other ingredients and components.Examples of other ingredients that may be included within thecomposition are emollients, sterols or sterol derivatives, natural andsynthetic fats or oils, viscosity enhancers, rheology modifiers,polyols, surfactants, alcohols, esters, silicones, clays, starch,cellulose, particulates, moisturizers, film formers, slip modifiers,surface modifiers, skin protectants, humectants, sunscreens, and thelike.

Pharmaceutical Compositions and/or Preparations:

Embodiments described herein further relate to pharmaceuticalcompositions and/or preparations comprising one or more of theaforementioned compounds of Formula I and a carrier. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, salts, compositions, dosage forms, etc., which are—within thescope of sound medical judgment—suitable for use in contact with thetissues of human beings and/or other mammals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. In some aspects,“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government, or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in mammals (e.g.,animals), and more particularly, in humans.

The pharmaceutical compositions may be prepared by any suitable meansknown in the art. Examples of such compositions include those adaptedfor (a) topical application, e.g., articles (e.g., gauzes, pads, swabs,dressings), creams, ointments, gels, lotions, etc.; (b) parenteraladministration, e.g., subcutaneous, intramuscular or intravenousinjection as a sterile solution or suspension; (c) oral administration,external application (e.g. drenches including aqueous and non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pelletsfor admixture with feedstuffs, pastes for application to the tongue,etc.

In certain embodiments, the pharmaceutical compositions may comprise oneor more antibiotic agents. As used herein, the term “antibiotic” or“antimicrobial agent” refers to a substance that inhibits the growth ofor destroys microorganisms. Preferably, the antibiotic is useful incurbing the virulence of an infectious agent and/or treating aninfectious disease. Antibiotic also refers to semi-synthetic substanceswherein a natural form produced by a microorganism, e.g., yeast orfungus is structurally modified.

Preferably, the antibiotic is selected from the group consisting ofβ-lactams (including, β-lactamase inhibitors and cephalosporins),fluoroquinolones, aminoglycosides, tetracyclines and/or glycylcyclinesand/or polymyxins. Any combination of antimicrobial agents may also beemployed, e.g., at least one β-lactam and at least one fluoroquinolone;at least one aminoglycoside and one cephalosporin; at least one β-lactamand one β-lactamase inhibitor, optionally together with anaminoglycoside, etc.

As used herein, the term “β-lactam” inhibitor includes natural andsemi-synthetic penicillins and penicillin derivatives, e.g., benzathinepenicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin(penicillin V), procaine penicillin and oxacillin; methicillin,dicloxacillin and flucloxacillin; temocillin; amoxicillin andampicillin; azlocillin, carbenicillin, ticarcillin, mezlocillin andpiperacillin; biapenem, doripenem, ertapenem, imipenem, meropenem,panipenem and PZ-601; cephalexin, cephalothin, cefazolin, cefaclor,cefuroxime, cefamandole, cefotetan, cefoxitin, cefotaxime, andcefpodoxime; cefepime and cefpirome; cefadroxil, cefixime, cefprozil,cephalexin, cephalothin, cefuroxime, cefamandole, cefepime andcefpirome; cefoxitin, cefotetan, cefmetazole and flomoxef; tigemonam,nocardicin A and tabtoxin; clavulanic acid, moxalactam and flomoxef.Fluoroquinolones include, ciprofloxacin, garenoxacin, gatifloxacin,gemifloxacin, levofloxacin, and moxifloxacin. Aminoglycosides include,for e.g., kanamycin, amikacin, tobramycin, dibekacin, gentamicin,sisomicin, netilmicin, neomycin B, neomycin C, neomycin E (paromomycin)and streptomycin, including, synthetic derivatives clarithromycin andazithromycin. Tetracyclines include naturally-occurring compounds (e.g.,tetracycline, chlortetracycline, oxytetracycline, demeclocycline) orsemisynthetic agents (e.g., lymecycline, meclocycline, methacycline,minocycline, rolitetracycline). Glycylcyclines (e.g.,minocycline/tigecycline) are derived from tetracyclines. Polymyxinsinclude, e.g., polymyxin B and polymyxin E (colistin).

In certain embodiments, the compositions may contain an antibiotic at aconcentration of 0.1 mg/mL, 0.5 mg/L, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL,12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, 25 mg/mL, 26mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 31 mg/mL, 32 mg/mL, 33mg/mL, 34 mg/mL, 35 mg/mL, 36 mg/mL, 37 mg/mL, 38 mg/mL, 39 mg/mL, 40mg/mL, 41 mg/mL, 42 mg/mL, 43 mg/mL 44 mg/mL, 45 mg/mL, 50 mg/mL, 60mg/mL, 70 mg/mL, 80 mg/m, 90 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250mg/mL, 300 mg/mL, 400 mg/mL, 500 mg/mL, or more. For example, imipenemand ertapenem may be used in the concentrations of 50, 30, 20, 15, 10, 5and 1 mg/mL.

Wound Dressings:

Disclosed herein, in certain embodiments, are wound dressings comprisingwound dressing materials as described herein, e.g., compounds of FormulaI. In some embodiments, the wound dressings consist essentially of thewound dressing materials as described herein, e.g., a compound ofFormula I.

In one embodiment, the wound dressing disclosed herein arebiocompatible, biodegradable, non-immunogenic and readily commerciallyavailable.

In one embodiment, the compounds of Formula I are provided in the formof particles, such as fiber particles or powder particles, optionallycontaining a medicament. In particular, the materials preferably containPG fibers.

The compositions may preferably comprise an intimate mixture of thedressing material and other compounds. For instance, in one embodiment,the intimate mixture comprises a mixed solution or dispersion of thedressing material and a suitable vehicle, such as a solvent, or a solidcomposition produced by removing solvent from such a solution ordispersion. Under this embodiment, the dressing material makes up atleast 5%, more preferably at least 10%, 20%, 30%, 50%, 75%, 90% orgreater % by weight of the material. In certain preferred embodiments,the material consists essentially of the dressing material.

Other components of the material may include 0-25% by weight, forexample from about 1 to about 20% by weight, of one or more otherbiocompatible polysaccharides, for example alginates such as sodiumalginate or calcium alginate, starch derivatives such as sodium starchglycolate, cellulose derivatives such as methyl cellulose orcarboxymethyl cellulose, or glycosaminoglycans such as hyaluronic acidor its salts, chondroitin sulfate or heparan sulfate. The materials mayalso comprise up to about 25% by weight, for example from about 1 toabout 20% by weight, of one or more structural proteins selected fromthe group consisting of fibronectin, fibrin, laminin, elastin, collagenand mixtures thereof. Preferably the protein comprises collagen, andmore preferably it consists essentially of collagen. The materials mayalso comprise up to about 20% by weight, preferably from about 2% toabout 10% by weight of water. The materials may also contain 0-40% byweight, for example from about 5 to about 25% by weight, of aplasticizer, preferably a polyhydric alcohol such as glycerol orsorbitol.

In certain embodiments, the materials may also comprise up to about 10%by weight, for example from about 0.01 to about 5% by weight, typicallyfrom about 0.1 to about 2% by weight of one or more therapeutic woundhealing agents, such as non-steroidal anti-inflammatory drugs (e.g.,acetaminophen), steroids, local anesthetics, antimicrobial agents, orgrowth factors (e.g., fibroblast growth factor or platelet derivedgrowth factor). The antimicrobial agent may, for example, comprise anantiseptic, an antibiotic, or mixtures thereof. Preferred antibioticsinclude tetracycline, penicillins, terramycins, erythromycin,bacitracin, neomycin, polymycin B, mupirocin, clindamycin and mixturesthereof. Preferred antiseptics include silver, including colloidalsilver, silver salts including salts of one or more of the anionicpolymers making up the material, silver sulfadiazine, chlorhexidine,povidone iodine, triclosan, sucralfate, quaternary ammonium salts andmixtures thereof. These medicated wound dressing materials according tothe disclosed technology provide sustained release of the therapeuticagents as the wound dressing material breaks down in use.

All of the above percentages are on a dry weight basis. Preferably, theweight ratio of the wound dressing material to other auxiliary agentsand materials is from about 1:99 to about 99:1. More preferably, theweight ratio is in the range about 1:9 to about 9:1, more preferably itis in the range about 4:1 to about 1:4, still more preferably in therange about 2:1 to about 1:2.

The material may be in any convenient form, such as a powder,microspheres, flakes, a mat or a film.

In certain embodiments, the material is in the form of a semisolid orgel ointment for topical application.

In certain embodiments, the material is in the form of a freeze-dried orsolvent-dried bioabsorbable sponge for application to a chronic wound.Preferably, the average pore size of the sponge is in the region of10-500 μm, more preferably about 100-300 μm. A suitable sponge has beenmade by freeze-drying or solvent drying an aqueous dispersion comprisingcompounds of Formula I, together with suitable therapeutic agents.

In yet other embodiments, the material is in the form of a flexiblefilm, which may be continuous or interrupted (e.g. perforated). Theflexible film preferably comprises a plasticizer to render it flexible,such as glycerol.

The ready availability of both gel forming polymers, e.g., cellulosederivatives, having a range of controllable properties means that theproperties of the compositions the disclosed technology can becontrolled to an exceptional degree. In particular, the rate ofbiological absorption, porosity and density of the materials can becontrolled.

In one embodiment, provided herein are wound dressing materials in sheetform, comprising an active layer of a composition comprising compoundsof Formula I. The active layer would normally be the wound contactinglayer in use, but in some embodiments it could be separated from thewound by a liquid-permeable top sheet. In one embodiment, the area ofthe active layer is from about 1 cm² to about 400 cm², particularly fromabout 4 cm² to about 100 cm².

In another embodiment, the wound dressing material further comprises abacking sheet extending over the active layer opposite to the woundfacing side of the active layer. Preferably, the backing sheet is largerthan the active layer such that a marginal region of width 1 mm to 50mm, preferably 5 mm to 20 mm extends around the active layer to form aso-called island dressing. In such cases, the backing sheet ispreferably coated with a pressure sensitive medical grade adhesive in atleast its marginal region.

In embodiments wherein the dressing material comprises a backing sheet,the back sheet is substantially liquid-impermeable. In anotherembodiment, the backing sheet is semipermeable, e.g., the backing sheetis preferably permeable to water vapor, but not permeable to liquidwater or wound exudate. Preferably, the backing sheet is alsomicroorganism-impermeable. Suitable continuous conformable backingsheets will preferably have a moisture vapor transmission rate (MVTR) ofthe backing sheet alone of 300 to 5000 g/m²/24 hrs, preferably 500 to2000 g/m²/24 hrs at 37.5° C. at 100% to 10% relative humiditydifference. The backing sheet thickness is preferably in the range of 10to 1000 micrometers, more preferably 100 to 500 micrometers.

Suitable polymers for forming the backing sheet include polyurethanesand poly alkoxyalkyl acrylates and methacrylates. Preferably, thebacking sheet comprises a continuous layer of a high density blockedpolyurethane foam that is predominantly closed-cell. A suitable backingsheet material is a polyurethane film.

In wound dressings comprising a backing layer comprising an adhesive,the adhesive layer should be moisture vapor transmitting and/orpatterned to allow passage of water vapor. The adhesive layer ispreferably a continuous moisture vapor transmitting, pressure-sensitiveadhesive layer of the type conventionally used for island-type wounddressings, for example, a pressure sensitive adhesive based on acrylateester copolymers, polyvinyl ethyl ether and polyurethane.Polyurethane-based pressure sensitive adhesives may be selectively used.

In another embodiment, the dressing may comprise further layers of amultilayer absorbent article may be built up between the active layerand the protective sheet. For example, these layers may comprise anapertured plastic film to provide support for the active layer in use,in which case the apertures in the film are preferably aligned inregister with the apertures in the hydrogel layer.

Still further, in other embodiments, the dressing may comprise anabsorbent layer between the active layer and the protective sheet,especially if the dressing is for use on exuding wounds. The optionalabsorbent layer may be any of the layers conventionally used forabsorbing wound fluids, serum or blood in the wound healing art,including gauzes, nonwoven fabrics, superabsorbents, hydrogels andmixtures thereof. Preferably, the absorbent layer comprises a layer ofabsorbent foam, such as an open celled hydrophilic polyurethane foam. Inother embodiments, the absorbent layer may be a nonwoven fibrous web,for example a carded web of viscose staple fibers.

In certain embodiments, the wound dressing may be protected by aremovable cover sheet. The cover sheet is normally formed from flexiblethermoplastic material. Suitable materials include polyesters andpolyolefins. Preferably, the adhesive-facing surface of the cover sheetis a release surface. That is to say, a surface that is only weaklyadherent to the active layer and the adhesive on the backing sheet toassist peeling of the hydrogel layer from the cover sheet. For example,the cover sheet may be formed from a non-adherent plastic such as afluoropolymer, or it may be provided with a release coating such as asilicone or fluoropolymer release coating.

In one embodiment, the wound dressing is sterile and packaged in amicroorganism-impermeable container.

Kits:

In certain embodiments, the disclosed technology provides kitscomprising, in one or separate compartments, the compounds of Formula I,optionally together with an excipient, carrier or oil. The kits mayfurther comprise additional ingredients, e.g., gelling agents,emollients, surfactants, humectants, viscosity enhancers, emulsifiers,etc., in one or more compartments. The kits may optionally compriseinstructions for formulating an article for diagnosing, detecting ortreating wounds, e.g., chronic or infected wounds. The kits may alsocomprise instructions for using the components, either individually ortogether, in the treatment of wounds.

In a related embodiment, the disclosed technology provides kitscomprising a package and at least one absorbent article (describedabove) comprising the aforementioned compositions. Alternately, the kitsmay comprise the individual components separately, optionally togetherwith secondary information, useable in or with the package.

Other embodiments disclosed herein relate to the use of the compositionfor the preparation of a dressing for the treatment of a wound.Preferably, the wound is a chronic wound, for example a wound selectedfrom the group consisting of venous ulcers, decubitis ulcers anddiabetic ulcers.

Surfaces:

Embodiments of the disclosed technology further provide for surfacescomprising the aforementioned compounds of Formula I, wherein thereporter or peptide is oriented to permit binding to a partner, e.g., anenzyme. Preferably, the surface is a surface of a solid support.Numerous and varied solid supports are known to those in the art. Usefulsolid supports include natural polymeric carbohydrates and theirsynthetically modified, cross-linked or substituted derivatives, such asagar, agarose, cross-linked alginic acid, substituted and cross-linkedguar gums, cellulose esters, especially with nitric acid and carboxylicacids, mixed cellulose esters, and cellulose ethers; natural polymerscontaining nitrogen, such as proteins and derivatives, includingcross-linked or modified gelatins; natural hydrocarbon polymers, such aslatex and rubber; synthetic polymers which may be prepared with suitablyporous structures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer.

In one embodiment, the support is a well of an array plate, e.g., amicroarray. Methods for constructing such arrays are known in the art,e.g., Cao et al., Appl Environ Microbiol., 77(23): 8219-8225, 2011. Eachcompound of Formula I (or the peptide indicators alone) may be spottedin triplicate to eliminate irregular data due to physical defects in thearray.

Systems:

Embodiments of the disclosed technology further provide for diagnosticsystems comprising the aforementioned compositions and/or kits.

The various components of the diagnostic systems may be provided in avariety of forms. For example, the compounds of Formula I {e.g.,compounds containing peptide reporters) may be provided as a lyophilizedreagent. These lyophilized reagents may be pre-mixed beforelyophilization so that when reconstituted they form a complete mixturewith the proper ratio of each of the components ready for use in theassay. In addition, the diagnostic systems of the disclosed technologymay contain a reconstitution reagent for reconstituting the lyophilizedreagents of the kit.

Nucleic Acids

In one embodiment, included herein are nucleic acids encoding thefollowing peptides:

X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z, X_(y)AAP(V/F/A)X_(y)-Z orX_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Z orX_(y)N⁴N³N²N¹X_(y)-L-Z, wherein X, y, N1, N2, N3, N4, L and Z are each,as described above.

In one embodiment, included herein are nucleic acids encoding thefollowing peptide X_(y)UUUU_(y)-Z, wherein X, y, U and Z are eachdescribed above.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, “fragments” refers to those nucleicacid sequences which are greater than about 10 nucleotides in length,and most preferably are at least about 40 nucleotides, at least about100 nucleotides, or at least about 300 nucleotides in length.

Embodiments disclosed herein further relate to variants of theaforementioned polynucleotides.

In one embodiment, included herein are variants of aforementionednucleic acids which comprise, or alternatively consist of, a nucleotidesequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, orgreater % identity to, for example, the nucleic acids encoding thefollowing peptides: X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Zor X_(y)N⁴N³N²N¹X_(y)-L-Z, wherein X, y, NI, N2, N3, N4, L and Z areeach, as described above.

In one embodiment, included herein are variants of aforementionednucleic acids which comprise, or alternatively consist of, a nucleotidesequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, orgreater % identity to, for example, the nucleic acids encoding thefollowing peptide X_(y)UUUU_(y)-Z, wherein X, y, U and Z are eachdescribed above.

One skilled in the art can use routine software, e.g., Three-to-OneSequence Manipulation Suite (which generates three potential nucleicacid sequences for each inputted polypeptide sequence), to arrive at theencoding nucleic acid sequences. The Three-to-One software is availablefreely from bioinformatics(dot)org.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (LASERGENE softwarepackage, DNASTAR). The MEGALIGN program can create alignments betweentwo or more sequences according to different methods, e.g., the ClustalMethod. (Higgins et al., Gene 73:237-244, 1988). The CLUSTAL algorithmgroups sequences into clusters by examining the distances between allpairs. The clusters are aligned pairwise and then in groups. Thepercentage similarity between two amino acid sequences, e.g., sequence Aand sequence B, is calculated by dividing the length of sequence A,minus the number of gap residues in sequence A, minus the number of gapresidues in sequence B, into the sum of the residue matches betweensequence A and sequence B, times one hundred. Gaps of low or of nohomology between the two amino acid sequences are not included indetermining percentage similarity. Percent identity between nucleic acidsequences can also be calculated by the Clustal Method, or by othermethods known in the art, such as the Jotun Hein Method. (See, e.g.,Hein et al. (1990) Methods Enzymol. 183:626-645.) Identity betweensequences can also be determined by other methods known in the art,e.g., by varying hybridization conditions.

In another embodiment, included herein are variant polynucleotides whichhybridize to one or more nucleic acid molecules under stringenthybridization conditions or lower stringency conditions.“Hybridization,” as the term is used herein, refers to any process bywhich a strand of nucleic acid bonds with a complementary strand throughbase pairing. For example, hybridization under high stringencyconditions could occur in about 50% formamide at about 37° C. to 42° C.Hybridization could occur under reduced stringency conditions in about35% to 25%) formamide at about 30° C. to 35° C. In particular,hybridization could occur under high stringency conditions at 42° C. in50% formamide, 5 SSPE, 0.3% SDS, and 200 μg/ml sheared and denaturedsalmon sperm DNA. Hybridization could occur under reduced stringencyconditions as described above, but in 35% formamide at a reducedtemperature of 35° C. The temperature range corresponding to aparticular level of stringency can be further narrowed by calculatingthe purine to pyrimidine ratio of the nucleic acid of interest andadjusting the temperature accordingly. Variations on the above rangesand conditions are well known in the art.

The term “hybridization complex” as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution or formed between one nucleic acid sequencepresent in solution and another nucleic acid sequence immobilized on asolid support (e.g., paper, membranes, filters, chips, pins or glassslides, or any other appropriate substrate to which cells or theirnucleic acids have been fixed).

In another embodiment, included herein are variants which arepolynucleotide fragments of the aforementioned nucleic acids.

Also included herein are oligonucleotides, e.g., PCR primers, whichhybridize to one or more nucleic acids. The term “oligonucleotide,” asused herein, refers to a nucleic acid sequence of at least about 6nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides,and most preferably about 20 to 25 nucleotides, which can be used in PCRamplification or in a hybridization assay or microarray. As used herein,the term “oligonucleotide” is substantially equivalent to the terms“amplimers,” “primers,” “oligomers,” and “probes,” as these terms arecommonly defined in the art.

Also included herein are modified nucleic acids such as PNA. “Peptidenucleic acid” (PNA), as used herein, refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAand RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell. (See, e.g., Nielsen et al. (1993) AnticancerDrug Des. 8:53-63.).

Vectors

Also included herein are vectors which contain one or more of theaforementioned nucleic acids. In one embodiment, the vector comprises atleast one protein encoding nucleic acid, e.g., nucleic acids encodingthe polypeptide sequences for X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Zor X_(y)N⁴N³N²N¹X_(y)-L-Z or X_(y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z,and U have been described previously] or an enzyme-cleavable fragmentthereof and/or an immunogenic fragment thereof, in operable linkage withone or more additional sequences. The additional sequences may besynthetic in nature. The terms “operably associated” or “operablylinked,” as used herein, refer to functionally related nucleic acidsequences. A promoter is operably associated or operably linked with acoding sequence if the promoter controls the transcription of theencoded polypeptide. While operably associated or operably linkednucleic acid sequences can be contiguous and in reading frame, certaingenetic elements, e.g., repressor genes, are not contiguously linked tothe encoded polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

Codon Optimized Sequences

Included herein are codon-optimized sequences of the aforementionednucleic acid sequences and vectors. Codon optimization for expression ina host cell, e.g., bacteria such as E. coli or insect Hi5 cells, may beroutinely performed using Codon Optimization Tool (CodonOpt), availablefreely from Integrated DNA Technologies, Inc., Coralville, Iowa.

Host Cells

Included herein are host cells containing the aforementioned nucleicacid sequences and vectors. In one embodiment, the host cell is capableof recombinantly expressing the gene sequence contained in the vectorunder standard culture conditions to generate the polypeptide product,e.g., polypeptide sequences for X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Zor X_(y)N⁴N³N²N¹X_(y)-L-Z or X_(y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z,and U have been described previously] or an enzyme-cleavable fragmentthereof and/or an immunogenic fragment thereof. In one specificembodiment, the host cell is E. coli.

Polypeptides

In one embodiment, included herein are polypeptides comprising thefollowing amino acid sequences: X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Zor X_(y)N⁴N³N²N¹X_(y)-L-Z or X_(y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z,and U have been described previously].

In another embodiment, included herein are variants of aforementionedpolypeptides which comprise, or alternatively consist of, an amino acidsequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, orgreater % identity to, for example, the following polypeptide sequences:X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z, X_(y)AAP(V/F/A)X_(y)-Z orX_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Z or X_(y)N⁴N³N²N¹X_(y)-L-Zor X_(y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z, and U have been describedpreviously]. Particularly, the fragment comprises a minimal structuralmotif for the enzyme recognition site (R) for the enzymes describedherein, e.g., lysozyme, elastase, cathepsin G, MAO, proteinase 3C or acombination thereof. Alternately or additionally, the fragment peptidesare immunogenic molecules that can be recognized by antibodies orantigen-binding domains thereof.

Homologs

In another embodiment, included herein are homologs to theaforementioned peptides and polynucleotides. The term “homology,” asused herein, refers to a degree of complementarity. There may be partialhomology or complete homology. The word “identity” may substitute forthe word “homology.” A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to as “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or northern blot, solution hybridization, and the like) underconditions of reduced stringency. A substantially homologous sequence orhybridization probe will compete for and inhibit the binding of acompletely homologous sequence to the target sequence under conditionsof reduced stringency. This is not to say that conditions of reducedstringency are such that nonspecific binding is permitted, as reducedstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., a selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% homology or identity). In the absence of nonspecificbinding, the substantially homologous sequence or probe will nothybridize to the second non-complementary target sequence.

Mutants

In another embodiment, included herein are variant peptides comprising amutation in the core polypeptide sequence for X_(y)AAPX_(y)-Z,X_(y)AAPX_(y)-L-Z, X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z;X_(y)N⁴N³N²N¹X_(y)-Z or X_(y)N⁴N³N²N¹X_(y)-L-Z or X_(y)UUUU_(y)-Z[wherein X, y, N1-N4, L, Z, and U have been described previously] or anenzyme-cleavable fragment thereof.

In one embodiment, the mutation is a substitution, deletion, addition of1-3 amino acids. Preferably, the mutation does not change the enzymerecognition sites in the mutant peptides so formed. If the mutationresults in a change in the composition of the recognition site orcleavage site, then it is contemplated that the mutation is due to aconserved amino acid substitution,

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule. A “substitution,” as usedherein, refers to the replacement of one or more amino acids ornucleotides by different amino acids or nucleotides, respectively.

Antibodies

Embodiments disclosed herein further include antibodies which bindspecifically to one or more of the aforementioned immunogenic peptides.

In one embodiment, the antibodies bind to polypeptides comprising thefollowing amino acid sequences: X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Zor X_(y)N⁴N³N²N¹X_(y)-L-Z or X_(y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z,and U have been described previously]. In another embodiment, theantibodies bind to fragment of these polypeptides. Contemplated hereinare antigen-binding fragments of such antibodies, e.g., F(ab) domain,F(ab)₂ domains, scFv domains, including synthetically generatedantibodies (using, e.g., phase display technology).

In one embodiment, the antibodies bind to polypeptide sequences forX_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z, X_(y)AAP(V/F/A)X_(y)-Z orX_(y)AAP(V/F/A)X_(y)-L-Z; X_(Y)N⁴N³N²N^(X)X_(Y)-Z orX_(Y)N⁴N³N²N^(X)X_(Y)-L-Z or X_(y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z,and U have been described previously] or an enzyme-cleavable fragmentthereof and/or an immunogenic fragment thereof. Contemplated herein areantigen-binding fragments of such antibodies, e.g., F(ab) domain, F(ab)₂domains, scFv domains, including synthetically generated antibodies(using, e.g., phase display technology).

Purified Molecules

Included herein are purified biomolecules, e.g., nucleic acids,proteins, peptides, and/or antibody molecules, including, conjugatesthereof. The term “substantially purified,” as used herein, refers tonucleic acids, amino acids or antibodies that are removed from theirnatural environment and are isolated or separated, and are at leastabout 60% free, preferably about 75% free, and most preferably about 90%free from other components with which they are naturally associated.

In embodiments described herein, the biomolecules may be altered bycombining with various components of the chemical entities, e.g., anchorregion and/or indicator region, such that their form and/orfunctionality is significantly changed compared to any naturalcounterparts.

Methods of Making Compounds of Formula I:

Embodiments provided herein further relate to methods of makingcompounds of Formula I, including precursors thereof. The term“precursor” includes any compound which is employed as a reactant togenerate an intermediary or a final product.

In one embodiment, provided herein is a method of making a compound ofFormula I comprising the structure A-R-1, wherein, A is an anchor asdescribed above and I is an indicator as described above, comprising,conjugating the anchor with the indicator molecule, e.g., via covalentbond. In one embodiment, the anchor or the indicator may comprise arecognition site (R) for a wound-specific marker, e.g., a wound-specificenzyme such as a hydrolase, and more specifically a protease orglycosidase, as described before. Under this embodiment, the substratefor the wound-specific marker comprises, for example, a hydrolysablesubstrate, e.g., an amino acid, a sugar, a peptide, a polysaccharide, anucleic acid, a lipid, a lactam or a combination thereof.

In one embodiment, the anchor is conjugated to the reporter molecule viaa peptide linkage, a glycosidic linkage, an amide linkage, an esterlinkage, an ether linkage, an anhydride linkage or a similar linkage. Asused herein, a “peptide bond” is formed by the condensation reactionbetween two amino acids, wherein the acid moiety of one reacts with theamino moiety of the other to produce a peptide bond (—CO— H—) betweenthe two amino acids. In one embodiment, the peptide bond is cleaved witha wound-specific protease, e.g., elastase, cathepsin G, proteinase C, orMAO, or a combination thereof. As used herein, a “glycosidic bond” isformed between the hemiacetal or hemiketal group of a saccharide (or amolecule derived from a saccharide) and the hydroxyl group of somecompound such as an alcohol. In one embodiment, the peptide bond iscleaved with a wound-specific glycosidase, e.g., lysozyme. A lactam bondis formed between an amide and a lactone and is found in the corestructure of many antibiotics. In one embodiment, the lactam bond iscleaved by a beta-lactamase.

Methods for conjugating reactive moieties to generate glycosidic,peptide, ester, oxyester, amide, amido, oxyamido, ether, sulfonyl,sulfinyl, sulfonamide, or other linkages such as alkoxy, alkylthio,alkylamino, etc. are known in the art and are further described in theexamples.

In another embodiment, provided herein is a method of making a compoundof Formula I comprising the structure A-I, wherein, A and I are each, asdescribed previously.

In one embodiment, the A is conjugated to the I via a glycosidic linkageor a peptide linkage.

In another embodiment, the A is conjugated to the I via a hydrophilic orhydrophobic linkage.

In one embodiment, the compound of Formula I having the structure A-I issynthesized by first conjugating the anchor region A with the indicatorregion I to generate the compound of Formula I.

In another embodiment, the indicator is first synthesized via geneticrecombinant technology, e.g., expressing a nucleic acid encoding theindicator region in a suitable host cell, and combining the indicatorwith the anchor region. Under this embodiment, in one instance, theindicator region is designed to contain nucleic acid sequences whichbind to the anchor region, e.g., hydrophilically or hydrophobically. Onerepresentative example of a hydrophilic interaction comprises use of ananchor containing polar groups, e.g., partially carboxylated sugar orpeptidoglycan, which interacts with polar amino acids in the indicatormolecule. Another representative example of a hydrophobic interactioncomprises use of an anchor containing non-polar groups, e.g., amidatedor esterified side chains (or a derivative thereof), which interactswith hydrophobic amino acid residues in the indicator molecule.

In one embodiment, peptide indicators, e.g., polypeptides comprising thefollowing amino acid sequences: X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Zor X_(y)N⁴N³N²N¹X_(y)-L-Z or X_(y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z,and U have been described previously], (including variant polypeptides)may be synthesized via host-cell expression systems. Such a method maycomprise, for example, generating a construct encoding one or more ofthe aforementioned polypeptides or variants, placing said construct in asuitable vector, e.g., plasmid vector or baculovirus vector,transfecting a host cell, e.g., E coli or insect Hi5 cells, with thevector; culturing the host cells under suitable conditions to allowexpression of said vector; and optionally purifying the expressedpolypeptide from the culture.

In another embodiment, peptide indicators, e.g., polypeptides comprisingthe following amino acid sequences: X_(y)AAPX_(y)-Z, X_(y)AAPX_(y)-L-Z,X_(y)AAP(V/F/A)X_(y)-Z or X_(y)AAP(V/F/A)X_(y)-L-Z; X_(y)N⁴N³N²N¹X_(y)-Zor X_(y)N⁴N³N²N¹X_(y)-L-Z or X_(Y)UUUU_(y)-Z [wherein X, y, N1-N4, L, Z,and U have been described previously] (including variant polypeptides)may be synthesized using solid-phase peptide synthesis (see, Merrifieldet al., J. Am. Chem. Soc. 85 (14): 2149-2154).

Still further, the compound of Formula I having the structure A-R—I maybe synthesized in a single reaction chamber or multiple reactionchambers.

Diagnostic and Therapeutic Methods:

In one embodiment, the compositions, dressing materials, articles, kitsand systems described herein are useful in diagnosing or treatingwounds, particularly chronic or infected wounds. Although any type ofwound may be diagnosed and/or treated, the embodiments are particularlysuitable for diagnosing and treating wounds that exude wound fluid. Forexample, the wound may be a chronic or acute wound. Representativeexamples of chronic wounds include, e.g., venous ulcers, pressure sores,decubitis ulcers, diabetic ulcers and chronic ulcers of unknownaetiology. Representative examples of acute wounds include, e.g., acutetraumatic laceration, perhaps resulting from an intentional operativeincision.

As used herein, the term “a wound fluid” refers to any wound exudate orother fluid (suitably substantially not including blood) that is presentat the surface of the wound, or that is removed from the wound surfaceby aspiration, absorption or washing. The determining, measuring orquantifying is suitably carried out on wound fluid that has been removedfrom the body of the patient, but can also be performed on wound fluidin situ. The term “wound fluid” does not normally refer to blood ortissue plasma remote from the wound site. The wound fluid is mammalianwound fluid, suitably human wound fluid.

In one embodiment, the diagnostic method comprises contacting a woundwith at least one composition comprising a compound of Formula I orFormula II, a dressing material comprising such compounds, articlecomprising such materials or compounds, kits comprising such materialsor compounds, or a system comprising such materials or compoundsdescribed herein; and measuring a parameter associated with the wound.In a specific embodiment, the parameter being measured is a level oractivity of a wound-specific hydrolase. Particularly, the parameterbeing measured is the activity of the hydrolase.

In the aforementioned embodiments, the measurement may either be made insitu or ex situ. As used herein, the term “in situ” refers to processes,events, objects, or components that are present or take place within thecontext of the system or device, including, the surrounding environment,for example, the biological material with which the composition,article, system or device is in contact with. As an example, an in situreaction may refer to the reaction of the various components present inthe device (e.g., compound of Formula I or Formula II), including,components provided by the human skin tissue (e.g., wound exudatecontaining the enzyme). The term is contrasted with ex situ, whichrefers to outside of the environment.

In a second embodiment, the measurement is performed ex situ, e.g.,removing the fluid from the wound for analysis in the apparatus ordevice of the disclosed technology.

Suitably, the measurement is made in situ.

In one diagnostic embodiment, the method comprising determining a levelof a reporter, e.g., a product of a substrate acted upon by awound-specific enzyme. More specifically, the method comprisesdetermining a level of a hydrolase enzyme product. As used herein, theterm “determining” includes measuring a numerical value of the activityor level of said hydrolase; establishing if the activity or level fallsabove or below a predetermined range; and/or comparing the numericalvalue of activity or level with a control standard. The control standardmay comprise determining a level or activity of the hydrolase in abiopsy material obtained from an unwounded site or from a healthysubject.

In one specific embodiment, the term “determining” comprises measuringthe parameter {e.g., activity or level) of at least one wound specificenzyme selected from lipase, esterase, peroxidase, oxidase, glycosidase,glucuronidase, glucosidase, galactosidase, napsin (aspartyl protease),glucosylceramidase glucuronidase, palmitoyl protein thioesterase,Cathepsins A, B, D, G, L, S, Z; acid ceramidase, lactoferrin (LF),lysozyme, myeloperoxidase (MPO), elastase, cathepsins, and proteinase-3or a combination thereof; establishing if said parameter exceeds a firstpredetermined threshold; and/or comparing the numerical value ofparameter with a control standard. The control standard may comprisedetermining a parameter of the protease in a biopsy material obtainedfrom an unwounded site or from a healthy subject. In relatedembodiments, the term “determining” comprises establishing whether aweighted average (weighted sum) of the parameters associated with aplurality of the aforementioned proteases exceeds a predeterminedthreshold value for said weighted average.

In one particular embodiment, the parameter is activity level of theanalyte {e.g. a protease) in a wound fluid. Typically, the activity ofan individual analyte is expressed in terms units/mL.

In another embodiment, the parameter is the level of the analyte {e.g.,protease) in a wound fluid. Typically, the term amount is alsoindicative of the activity of a particular analyte.

When used herein, the term “combined amount” or “combined activity”refers to a single numerical value that results from the application ofa mathematical function to a plurality of values, for example thoseamounts obtained for a number of individual analytes. For example, theterm “combined amount” or “combined activity” may refer to the sum orproduct of a group of individual values. Typically, the term “combinedamount” or “combined activity” relates to the sum of a group ofindividual values. For example, in suitable embodiments, the amount ofelastase refers to elastase-like activity (e.g., U/mL) and the amount ofmetalloproteinase (MMP) refers to total concentration of the respectiveanalyte (e.g., in ng/mL).

When used herein, the term “quantifying” refers to measuring an absolutenumerical quantity of a particular analyte(s) or substrate(s) in asample, within the margins of experimental error.

The term “marker” or “analyte” refers to any chemical entity that isidentified or determined using the apparatus, devices, kits or methodsdefined herein. The markers or analytes determined or identified by theapparatus, devices, kits or methods of the disclosed technology arecleaved products of the aforementioned enzymes.

When used herein, the term “predetermined range” refers to a data rangeor profile that the skilled person would understand is indicative of aparticular sub-class of patient. For instance, the predetermined rangemay be a data range or profile that is typical of a wound that wouldrespond well to a particular wound treatment, such as antibiotictherapy. Alternatively, the predetermined range may suitably refer to adata range that is typical of a wound that would not respond well to aparticular wound treatment, such as antibiotic therapy.

When used herein, the term “predetermined threshold” refers to a minimumlevel that the skilled person would determine is indicative of anon-healing wound based on statistical analysis of levels determined forknown healing and non-healing wounds, for example as explained furtherabove. For the test to be clinically useful, the threshold should be setat an appropriate level so that non-healing wounds with high proteaseactivity are correctly identified. Increasing the threshold willincrease the chance of only non-healing wounds being over the threshold.However, if the threshold is too high, wounds that are non-healing dueto a high level of proteases would not be identified and clinically thiswould mean they would not receive the required protease modulatingtreatment.

When used herein, the term “control standard” or “control” refers to adata set or profile that can be used as a reference or comparison inorder to define or normalize another data point or set of data. Forinstance, the term “control” or “control standard” may be data set orprofile that is indicative of a particular sub-class of patient.Suitably, the control standard may be a data set or profile indicativeof healing or non-healing wound status.

Suitably, in other aspects or embodiments of the disclosed technology,the “control” or “control standard” can be a data set or profile thatcan be used as a comparative tool to allow a skilled person to determinewhether a wound is likely to be responsive or non-responsive to a woundtreatment, such as antibiotic therapy. In one embodiment, the controlstandard is a data set or profile indicative of a patient that does notrespond well to wound treatment. Typically, the control standard is adata set or profile indicative of a patient that responds well to woundtreatment. Patients that tend to respond well to wound treatment asdisclosed herein exhibit lower combined amount or activity of hydrolasesthan patients that tend not to respond well to the treatment. Forexample, patients that tend to respond well to wound treatment asdisclosed herein exhibit lower combined amounts of at least onewound-specific hydrolase.

In one embodiment, the threshold human neutrophil elastase activity isabout 5 U/mL to about 30 U/mL, including all values in between, e.g.,about 6 U/mL, about 7 U/mL, about 8 U/mL, about 9 U/mL, about 10 U/mL,about 11 U/mL, about 12 U/mL, about 13 U/mL, about 14 U/mL, about 15U/mL, about 16 U/mL, about 17 U/mL, about 18 U/mL, about 19 U/mL, about20 U/mL, about 21 U/mL, about 22 U/mL, about 23 U/mL, about 24 U/mL,about 25 U/mL, or more, indicate chronic wound infection.

In one specific embodiment, the threshold human neutrophil elastaseactivity levels of at least 9.6 indicate chronic wound infection. Insome embodiments, human neutrophil elastase activity levels of at least22.9 U/mL indicate chronic wound infection.

In one embodiment, the threshold lysozyme activity levels of about 1000U/mL to about 10000 U/mL, including all values in between, e.g., about1100 U/mL, about 1200 U/mL, about 1300 U/mL, about 1400 U/mL, about 1500U/mL, about 1600 U/mL, about 1700 U/mL, about 1800 U/mL, about 1900U/mL, about 2000 U/mL, about 2100 U/mL, about 2200 U/mL, about 2300U/mL, about 2400 U/mL, about 2500 U/mL, about 2600 U/mL, about 2700U/mL, about 2800 U/mL, about 2900 U/mL, about 3000 U/mL, about 3250U/mL, about 3500 U/mL, about 3750 U/mL, about 4000 U/mL, about 4250U/mL, about 4500 U/mL, about 4750 U/mL, about 5000 U/mL, about 5250U/mL, about 5500 U/mL, about 5750 U/mL, about 6000 U/mL, or more,indicate chronic wound infection. In one specific embodiment, lysozymeactivity levels of at least 4800 U/mL indicate chronic wound infection.

In one embodiment, the threshold cathepsin G activity levels of about 10U/mL to about 100 U/mL, including all values in between, e.g., about 15U/mL, about 20 U/mL, about 25 U/mL, about 30 U/mL, about 35 U/mL, about40 U/mL, about 45 U/mL, about 50 U/mL, about 55 U/mL, about 60 U/mL,about 65 U/mL, about 70 U/mL, about 75 U/mL, about 80 U/mL, about 85U/mL, about 90 U/mL, about 95 U/mL, about 100 U/mL, about 110 U/mL,about 120 U/mL, or more, indicate chronic wound infection. In someembodiments, cathepsin G activity levels of at least 50 U/mL, at least40 U/mL, at least 30 U/mL, at least 20 U/mL, at least 15 U/mL or atleast 10 U/mL indicates chronic wound infection.

Embodiments disclosed herein further relate to treatment of chronic orinfected wounds using the compositions, materials, articles, dressings,kits and/or systems described herein. The therapeutic embodimentincludes, contacting a composition, material, article, dressing, kit,system or devices of the disclosed technology with a subject in needthereof. Optionally, the method may include determination of whether thesubject is responding to the treatment.

The skilled person would be able to easily identify whether wounds are“responsive to treatment” or not. In particular, the skilled person willreadily be able to determine the levels of the proteases identified inthe present claims that are predictive or indicative of a good responseor poor response to wound treatment, particularly to treatment withwound dressings comprising oxidized cellulose. The terms “responsive”and “responder(s)” as used herein refer to wounds that are considered torespond well to wound treatment, particularly to treatment with apharmacological agent, e.g., antibiotics. Similarly, “non-responsive”and “non-responder(s)” refers to wounds that are not considered torespond well to wound treatment, particularly to treatment with thepharmacological agent, e.g., antibiotics. For instance, patients whoexhibit better than 50% wound closure after 4 weeks of wound treatmentare considered to be responsive to said treatment.

In certain embodiments, a patient may be simultaneously diagnosed andtreated with the compositions, articles, systems, or devices describedherein. When used herein, the term “simultaneously” means performing thestated objectives, e.g., diagnosis and treatment, together.

In certain embodiments, a patient may be sequentially diagnosed andtreated with the compositions, articles, systems, or devices describedherein. When used herein, the term “sequentially” means the statedobjectives, e.g., diagnosis and treatment, are temporally or spatiallyseparated, e.g., diagnosis prior to treatment or diagnosis followingtreatment or a combination thereof, e.g., 1^(st)diagnosis==>treatment==>2^(nd) diagnosis.

Embodiments described herein further enable a care giver or a patient todetermine quickly and reliably whether a wound is likely to benon-healing, and to select an appropriate therapy based on thisdetermination. For example, non-healing wounds may require theapplication of special wound dressings such as wound dressingscomprising specific therapeutic agents, to promote healing. Accordingly,embodiments described herein further provide methods of treatment of awound, e.g., chronic or infected wounds, comprising determining whethera wound is healing or non-healing, followed by applying a wound dressingcomprising a therapeutic agent to the wound if it is non-healing.

Embodiments described herein provide methods and assays for diagnosis ordetection of infected wounds. The methods are suitable for the detectionof bacterial infectious agents. In one embodiment, the wounds areinfected with gram-negative bacteria. Typical gram-negative bacteriainclude proteobacteria such as E. coli, Salmonella, Pseudomonas, andHelicobacter, and cyanobacteria. When classified in connection withmedicine, they include Pseudomonas aeruginosa and Hemophilus influenzaecausing the disturbance of the respiratory system, Escherichia coli andProteus mirabilis causing the disturbance of the urinary system, andHelicobacter pylori and Bacillus Gaertner causing the disturbance of thealimentary system and micrococci such as Neisseria meningitidis,Moraxella catarrhalis, and Neisseria gonorrhea.

In another embodiment, the wounds are infected with gram-positivebacteria. By “gram-positive bacteria” is meant a bacterium or bacteriathat contain(s) teichoic acid {e.g., lipoteichoic acid and/or wallteichoic acid), or a functionally equivalent glycopolymer (e.g., arhamnopolysaccharide, teichuronic acid, arabinogalactan, lipomannan, andlipoarabinomannan) in its cell wall. Non-limiting examples offunctionally equivalent glycopolymers are described in Weidenmaier etal, Nature, 6:276-287, 2008.

The bacteria include pathogenic bacteria that infect mammalian hosts{e.g., bovine, murine, equine, primate, feline, canine, and humanhosts). Examples of such pathogenic bacteria include, e.g., members of abacterial species such as Bacteroides, Clostridium. Streptococcus,Staphylococcus, Pseudomonas, Haemophilus, Legionella, Mycobacterium,Escherchia, Salmonella, Shigella, Vibrio, or Listeria. Some clinicallyrelevant examples of pathogenic bacteria that cause disease in a humanhost include, but are not limited to, Bacillus anthracis, Bacilluscereus, Bordetella pertussis, Borrelia burgdorfen, Brucella aborus,Brucella canis, Brucella melitensis, Brucella suis, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Coryne bacterium diphtheriae, Enterococcus faecalis,vancomycin-resistant Enterococcus faecalis, Enterococcus faecium,Escherichia coli, enterotoxigenic Escherichia coli (ETEC),enteropathogenic Escherichia coli, E. coli 0157.H7, Francisellatularensis, Haemophilus influenzae, Helicobacter pylori, Legionellapneumophila, Leptospira interrogans, Listeria monocytogenes,Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitidis, Proteus, Pseudomonasaeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonellatyphimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcusepidermis, Staphylococcus saprophyticus, methicillin-resistantStaphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus(VSA), Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

In another embodiment, the infectious bacteria is selected from thegroup consisting of Clostridium difficile, Carbapenem-ResistantEnterobacteriaceae (CR-Klebsiella spp; CR-E. coli), and Neisseriagonorrhoeae. In another embodiment, the infectious bacteria is selectedfrom the group consisting of multidrug-resistant Acinetobacter,drug-resistant Campylobacter, extended spectrum P-Lactamase(ESBL)-producing enterobacteriaceae, vancomycin-resistant enterococcus,multidrug-resistant Pseudomonas aeruginosa, drug-resistant non-typhoidalSalmonella, drug-resistant Salmonella enterica serover Typhi,drug-resistant Shigella, methicillin-resistant Staphylococcus aureus(MRSA), drug-resistant Streptococcus pneumoniae, and drug-resistantTuberculosis. In another embodiment, the infectious bacteria is selectedfrom the group consisting of vancomycin-resistant Staphylococcus aureus,erythromycin-resistant Group A Streptococcus, clindamycin-ResistantGroup B Streptococcus.

In certain embodiments, the chronic or infected wounds are found in hostsubjects. Preferably, the hosts are mammals, e.g., a rodent, a human, alivestock animal, a companion animal, or a non-domesticated or wildanimal. In one embodiment, the subject may be a rodent, e.g. a mouse, arat, a guinea pig, etc. In another embodiment, the subject may be alivestock animal. Non-limiting examples of suitable livestock animalsmay include pigs, cows, horses, goats, sheep, llamas and alpacas. Instill another embodiment, the subject may be a companion animal.Non-limiting examples of companion animals may include pets such asdogs, cats, rabbits, and birds. In yet another embodiment, the subjectmay be a zoo animal. As used herein, a “zoo animal” refers to an animalthat may be found in a zoo. Such animals may include non-human primates,large cats, wolves, and bears. In an exemplary embodiment, the subjectis a human.

In one aspect, provided herein are methods of detecting levels of one ormore enzymes in a mammalian wound, the method comprising the steps of:(a) placing the wound dressing material described herein in contact withthe mammalian wound; (b) visually comparing the wound dressing materialin contact with the mammalian wound with one or more reference samples;and (c) obtaining a qualitative determination of the concentration ofreporter molecules in the wound dressing material in contact with themammalian wound.

Preferably, the diagnosis and treatment is conducted in situ.Embodiments described herein therefore allow diagnosis and treatment ofwounds in an easy, non-invasive manner. For instance, the diagnosis maybe made in real time and the treatment may be applied to the infectedwound or to the patient (systemically) and the progress of woundtreatment be monitored over real-time, e.g., dissipation of the signalgenerated by the reporter molecule due to wound-healing.

In another aspect, provided herein are methods of detecting proteaseactivity in wounds using a chemical entity, wherein the chemical entitycomprises one or more components selected from the group consisting of:an anchor region, an enzyme-labile or enzyme-reactive region, and anindicator region. In another aspect, the method compromises placingsubstrates for MPO, elastase, lysozyme, phospholipase, and catalase on asolid surface such that any reaction is visible to the eye. In anotheraspect, the method serves to assess a variety of body fluids includingwound, tear, vitreal, CSF, airway aspirates or sputum, synovial, blood,plasma, serum, urine, peritoneal, interstitial, subcutaneous, bile,intestinal or similar fluids, via contacting them with a materialcontaining the substrates and assessing the change of the substratesthereafter.

In another aspect, provided herein are methods of detecting infection inan airway, comprising contacting the chemical entities with the fluidfrom the infected organ either via specific sampling or via long-termcontact with a ventilation device.

EXAMPLES

The structures, materials, compositions, and methods described hereinare intended to be representative examples, and it will be understoodthat the scope of the disclosure is not limited by the scope of theexamples. Those skilled in the art will recognize that the embodimentsand disclosed technology may be practiced with variations on thedisclosed structures, materials, compositions and methods, and suchvariations are regarded as within the ambit of the disclosure.

All ionic compounds were handled and isolated as salts with variouscounter ions, depending on the last step and not further specified.

The “molecular weight” of poly-p-nitrophenylacrylates was determined asvirtual molecular mass by reaction with ethanolamine in DMF andquantitation of the released nitrophenol in IN NaOH at 405 nm. Virtualmolecular mass=g of material/reactive site. “H-RBB” refers to thereaction product of Remazol Brilliant Blue R® with ammonia (see example29), “RBB” to the respective radical the terminus, “H—” in compoundnames refers to hydrogen, not histidine; “normal conditions” means roomtemperature, atmospheric pressure, no protective means against humidityor oxygen; Peptide fragments are synthesized by conventional methodsknown to those skilled in the art; any peptides are described via theone-letter code; nevertheless single letters may also refer to heteroatoms; “CV” meansBis-(4-dimethylaminophenyl)-(4-[N-piperazino]phenyl)-carbenium ion, aderivative of crystal violet.

Example 1. Fmoc-AAPV-Indoxyl ester [SEQ ID NO: 2]

Fmoc-AAPV-OH [SEQ ID NO: 9] (43.47 g, 75.12 mmol) and CDI (14.62 g,90.19 mmol) were weighed directly into a 500 mL round bottom flask viapowder funnel. DCM (dry, 120 mL) was added directly to the flask,additional DCM (dry, 30 mL) was used to wash the powder funnel and wasadded to the flask as well. The mixture was stirred at RT under argonatmosphere for 15 min and then additional 45 min while passing argonthrough the mixture. Then the indoxyl was added in one portion using apowder funnel; funnel was washed with DCM (dry 10 mL) which was added tothe reaction mixture as well. Stirring at RT while passing argon throughthe reaction mixture was continued for 1 h, then additional DCM (dry, 20mL) was added and stirring at RT while passing argon through the mixturewas continued for 10 min. Afterwards the mixture was stirred at RT underargon atmosphere overnight. The reaction mixture was filtered viaBuchner-funnel with filter paper and filled into a separation funnel.The reaction flask was washed with DCM (30 mL), the wash-DCM wasfiltered via the Buchner funnel as well and filled into the separationfunnel, too. Water (350 mL) was added to the separation funnel; afterextraction the organic phase was collected. The separation funnel waswashes with acetone until no more colour was washed out, then it wasfilled with water completely and emptied again. The organic phase wasrefilled into the separation funnel and washed with water (350 mL) oncemore. After phase separation the organic phase was collected in a cleanErlenmeyer flask, dried (Na2SO4, 40.56 g), and concentrated to dryness.The crude product was suspended in diethy ether (600 mL) and stirred atRT for 2 h. The solid was filtered off via glass frit. Some solid stuckto the flask; it was dissolved in DCM (50 mL), concentrated to drynessand added to the rest of the solid. All the solid was resuspend indiethy ether (600 mL) and stirred at RT overnight. Afterwards the solidwas filtered off via glass frit again. Solid sticking to the flask wasdissolved in DCM (50 mL), concentrated to dryness and combined with therest of the solid. The product was kept at RT covered with a filterpaper with holes for 7 days and was then dried at the oil pump (RT, 2 h,0.008 mbar) to yield 41.4 g (79.4%) of the product.

Example 2. Fmoc-AAPV-5-Bromo-4-Chloro-Indoxyl ester [SEQ ID NO: 2]

Fmoc-AAPV-OH [SEQ ID NO: 9] (117 mg, 0.20 mmol) and CDI (39 mg, 0.24mmol) were dissolved in DCM (dry, 3 mL) and stirred at RT under argonatmosphere for 5 min. Then the mixture was stirred at RT for 5 min whilepassing argon through the mixture before 5-bromo-4-chloro-indoxyl (50mg, 0.20 mmol) was added in one portion. Passing argon through themixture was continued for 5 min, additional DCM (dry, 2 mL) was addedand the mixture was stirred at RT under argon atmosphere for 3.5 h.Afterwards the reaction mixture was concentrated to dryness and storedin the freezer under argon atmosphere for 18 d. The crude product waschromatographied: 15.8 g silica gel, eluent: 2% MeOH in DCM. A bluesolid (42 mg), still contaminated, was obtained. The product waschromatographed a second time: 13.7 g silica gel, eluent: startingeluent EtOAc/cyclohexane (3:1, 200 mL) the change to pure EtOAc. Analmost colorless solid (32 mg) was obtained. ESI-MS (positive): [M+H]⁺:805, [M+Na]⁺: 828, [M+K]⁺: 844

Example 3 Fmoc-AAAPV-Indoxylester [SEQ ID NO: 10]

Fmoc-AAAPV-OH [SEQ ID NO: 11] (361 mg, 0.56 mmol) and HOBt·H₂0 (127 mg,0.94 mmol) were dissolved in DCM (3 mL) at room temperature under argonatmosphere. DIPEA (190 μL, 0.90 mmol) and EDC·HCL (179 mg, 0.93 mmol)were added. The mixture was stirred under argon atmosphere at roomtemperature for 2 h. Then indoxyl (55 mg, 0.41 mmol) was added in oneportion while argon was passed through the mixture. passing argonthrough the reaction mixture was continued for 5 min more minutes; thenadditional DCM (2 mL) was added and the mixture was stirred under argonatmosphere at room temperature overnight. DCM (20 mL) was added and themixture was extracted with aq. sat. NaHCO₃ (for an acceleration of thephase separation, brine (5 mL) was added). The organic phase was washedwith brine (15 mL), dried (Na₂SO₄) and concentrated to dryness. Thecrude product was purified by column chromatography (15.0 g silica gel,40-63 μm) starting with DCM as the solvent. The column was eluted withDCM until all blue and pink color was washed down the column. Theneluent was changed to 5% MeOH in DCM. A colorless product (88 mg) and aslightly pink (contaminated) solid (49 mg) were collected.

Example 4. Fmoc-AAAPV-(N-acetyl)-indoxyl ester [SEQ ID NO: 10]

Fmoc-AAAPV-OH [SEQ ID NO: 11] (305 mg, 0.428 mmol) and HOB_(t)·H₂O (97.5mg, 0.47 mmol) were dissolved in DMF (2 mL) at room temperature underargon atmosphere. After 5 min, DIPEA (145.6 μL, 0.86 mmol) and EDCI·HCl(138 mg, 0.72 mmol) were added. The mixture was stirred at roomtemperature under argon atmosphere for 1 h (Mixture 1). In parallelindoxylacetate (50 mg, 0.285 mmol) was dissolved in DMF (2 mL) at roomtemperature under argon atmosphere. Argon was passed through thesolution for 5 min before NaOMe (15 mg, 0.270 mmol) was added. Passingargon through the mixture was continued for additional 20 min (Mixture2). Mixture 1 was added to Mixture 2 within a few seconds. A stream ofargon was purged through the reaction mixture for 2 h then the mixturewas stirred under argon atmosphere overnight. The mixture wasconcentrated to dryness, re-dissolved in EtOAc (50 mL), washed with aq.sat. NaHCO₃ (2×30 mL), water (1×20 mL) and brine (1×30 mL). The organicphase was dried (Na₂SO₄) and concentrated to dryness. The crude productwas purified by column chromatography (15.0 g silica gel, 40-63μττι,eluent: DCM) to yield 33 mg of the solid product. ESI-MS (positive):[M+H]⁺: 807; [M+Na]⁺: 829.

Example 5: Fmoc-AAAPV-3-Indolamide [SEQ ID NO: 10]

Fmoc-AAAPV-OH [SEQ ID NO: 11] and HOBt·H₂O were dissolved in DCM (dry.)in a 25 mL two necked round bottom flask at room temperature under argonatmosphere. DIPEA and EDCI*HCl were added. The mixture was stirred underargon atmosphere at room temperature for 1 h; then argon was passedthrough the mixture for 5 min. 1H-Indol-3-amine was added in one portionand passing argon through the mixture is continued for 5 min. Thenadditional DCM (dry, 2 mL).) was added and the mixture was stirred underargon atmosphere at room temperature for 2 h. DCM (25 mL) was added andthe mixture was extracted with sat. NaHCO₃-sol. (sat.); after extractionthe organic phase was washed with brine (20 mL). The combined aqueousphases were extracted with EtOAc (30 ml). (TLC of EtOAc and DCM phaseswith 5% MeOH in DCM: EtOAc: one spot; DCM. 3 spots). Both organic phaseswere dried (Na₂SO₄) and concentrated to dryness separately. The crudeproduct from the DCM phase was purified by column chromatographystarting with DCM as the solvent. The column was flushed with DCM untilall blue and pink color was washed down the column. Then eluent waschanged to 5% MeOH in DCM. Collected: 34 mg of a brown solid ESI-MS(positive): [M+H]⁺: 764; [M+Na]⁺:786.

Example 6: Fmoc-AAPV-4-methoxy-1-naphthol ester [SEQ ID NO: 2]

Fmoc-AAPV-OH [SEQ ID NO: 9] (169 mg, 0.29 mmol) and CDI (59 mg, 0.36mmol) were dissolved in DCM (dry, 3 mL) under argon atmosphere in at RT.After 10 min 4-methoxy-1-naphthol (50 mg, 0.29 mmol) was added and thesolution was stirred at RT for 45 min. DCM (30 mL) and water (20 ml)were added; after extraction the organic phase was dried with Na₂SO₄ andconcentrated to dryness. The crude product was purified by columnchromatography (eluent: 5% MeOH in DCM) to yield 58 mg of a beige solid.ESI-MS (positive): [M+H]⁺: 735; [M+Na]⁺: 757.

Example 7: Fmoc-AAPV-4-naphthol ester [SEQ ID NO: 2]

Fmoc-AAPV-OH [SEQ ID NO: 9] (241 mg, 0.42 mmol) and HOBt·H₂O (107 mg,0.70 mmol) were suspended in DCM (dry, 4 mL). After 5 min DIPEA (119 μL,0.70 mol) and EDCI*HC1 (134 mg, 0.7 mmol) were added. Then the1-Naphthol (50 mg, 0.35 mmol) was added in one portion, and the mixturewas stirred at RT under argon atmosphere over the weekend. DCM (20 mL)and water (20 mL) were added and the mixture was extracted. Since phaseseparation was very slow a brine solution. (10 mL) was added and themixture was extracted again. The organic phase was dried (Na₂SO₄) andconcentrated to dryness. The crude product was purified by columnchromatography (eluent: 5% MeOH in DCM) to yield 106 mg of the product.ESI-MS (positive): [M+H]⁺: 705; [M+Na]⁺: 727.

Example 8. Fmoc-AAPV-(2-Napthol) [SEQ ID NO: 2]

Synthesis: Fmoc-AAPV-OH [SEQ ID NO: 9] (241 mg, 0.42 mmol) and HOBt·H₂O(107 mg, 0.42 mmol) were suspended in dry DCM (3 mL) under argonatmosphere at RT. After 5 min DIPEA (119 μE, 0.7 mmol) and EDCPHC1 (134mg, 0.7 mmol) were added and the solution was stirred at RT for 45 min.Then the 2-Naphthol (50 mg, 0.35 mmol) was added in one portion, and themixture was stirred at RT under argon atmosphere for 2 h. DCM (30 mL)and water (30 ml) were added; after extraction the organic phase waswashed with a brine (30 mL), dried with Na₂SO₄ and concentrated todryness to yield 250 mg of an almost colorless solid. For purificationthe crude product was flashed over a short column of silica gel, eluent:5% MeOH in DCM. Yield: 168 mg of a colorless solid (68%). ESI-MS(positive) [M+H]⁺: 853.

Example 9: Methyl-3-Fmoc-AAPV-amide-4-aminobenzoate [SEQ ID NO: 2] andMethyl-3-amino-4-Fmoc-AAPV-amidobenzoate [SEQ ID NO: 2]

Fmoc-AAPV-OH [SEQ ID NO: 9] (214 mg, 0.37 mmol) and HOBt·H₂O (91 mg, 0.6mmol) were suspended in dry DCM (dry, 2 mL) under argon atmosphere atRT. After 5 min DIPEA (105 μL, 0.6 mmol) and EDCI*HC1 (115 mg, 0.6 mmol)were added and the solution was stirred at RT for 45 min. Thenmethyl-3,4-diaminobenzoate (50 mg, 0.3 mmol) was added in one portion,followed by additional DCM (dry, 1 mL), and the mixture was stirred atRT under argon atmosphere overnight. DCM (20 mL) and water (10 ml) wereadded; after extraction the organic phase is washed with sat. NaCl-sol.(10 mL), dried with Na₂SO₄ and concentrated to dryness. The crudeproduct was purified by column chromatography on silica gel. Eluent:started with 2% MeOH/DCM (approx. 120 mL) then changed to 5% MeOH inDCM. Yield: 97 mg containing a mixture of both isomers and 10 mg of onlyone of the isomers. ESI-MS (positive): [M+H]⁺: 727; [M+Na]⁴: 749.

Example 10. N-(1-[2-hydroxy]tetradecanyl) Fast blue RR(N-4-(1,3-Dimethoxy-2-N-benzoylamidophenyl)-N′-rac-(2-hydroxy-n-tetradecyl)-amine)

Fast Blue RR dye (7.3 mmol) was placed in a 3 necked round bottom flaskand was dissolved in chloroform (30 ml) at ambient temperature whilestirring (magnetic stirrer, 500 rpm). After 30 min. stirring1,2-epoxytetradecane (11 mmol) was added, followed by treatment withsulfuric acid (catalytic). Temperature was raised to reflux and stirringwas continued for 12 h. When in process control (ESI-MS (+p)) indicatedfull consumption of starting materials any solids were filtered off anddried in air stream. Several recrystallization steps (ethanol) werenecessary to obtain desired product in sufficiently pure form. Yield:600 mg. ESI-MS (positive mode) [M+H]⁺: 485.

Example 11. Fmoc-AAAPV-4diaminobenzoic acid) [SEQ ID NO: 10]

Fmoc-AAAPV-OH [SEQ ID NO: 11] (710 mg) was dissolved with DMF (5 mL).HOBt (210 mg) and DCC (240 mg) were added subsequently, and the mixturewas stirred for 15 min at RT. 3,4-Diaminobenzoic acid (160 mg) andpyridine (100 μL) were dissolved with DMF (1 mL) and added to reactionmixture with stirring. After 14 h, the solvent was evaporated, and theresidue was purified by silica gel chromatography withcyclohexane/DCM/methanol (4/2/1), containing 0.5% of formic acid, toyield 400 mg of product. ESI-MS (positive) [M+H]=784.

Example 12. Fmoc-AAPF-Indoxyl ester [SEQ ID NO: 12]

Fmoc-AAPF-OH [SEQ ID NO: 13] (994 mg, 1.59 mmol) and CDI (308 mg, 1.90mmol) were dissolved in dry DCM (4 mL). The mixture was stirred at RTunder argon atmosphere for 10 min and additional 20 min while passingargon through the mixture, followed by an add-on of additional DCM (dry,2 mL). Stirring while passing argon through the reaction mixture wascontinued for 2 min, then indoxyl (208 mg, 1.56 mmol) was added in oneportion. Stirring while passing argon through the solution was continuedfor 15 min, afterwards additional DCM (dry, 2 mL) was added and themixture was stirred at RT under argon atmosphere overnight. Reactionmixture was filled into a separation funnel, flask was washed with DCM(20 mL) and wash-DCM was added to separation funnel as well. The mixturewas washes with water (30 ml) and organic phase was collected togetherwith some slurry between organic and aqueous phase. Organic phase waswashed with water (30 mL) again, dried with Na₂SO₄ and concentrated todryness. The crude product was suspended in Et₂O (50 mL) and is stirredat RT for 90 min. Then the solid was filtered off and re-suspended infresh Et₂O (40 mL) and stirred at RT overnight. The solid was filteredoff and dried at oil pump to yield 444 mg (38%) of the product.

ESI-MS (positive): [M+H]⁺: 742; [M+Na]⁺: 764

Example 13: Fmoc-AAA-indoxyl ester

Fmoc-AAA-OH (103 mg, 0.52 mmol) and CDI (102 mg, 0.23 mmol) weredissolved in DCM (dry, 6 mL). The mixture was stirred at RT under argonatmosphere for 15 min and additional 10 min while passing argon throughit. Then indoxyl (30 mg, 0.23 mmol) was added in one portion. Stirringwhile passing argon through the mixture was continued for 10 min,afterwards additional DCM (dry, 2 mL) was added and the mixture wasstirred at RT under argon atmosphere overnight. Reaction mixture waswashed with water (a lot of solid sat between organic and aqueousphase). Organic phase was collected, washed with water once again, driedwith Na₂SO₄, and concentrated to dryness (only 33 mg).

The crude product was taken up in Et₂O (5 mL) and was shaken for 2 min.The liquid was decanted and 7 mg (5%) of the desired product could beobtained.

[M+Na]⁺: 591

Example 14: Fmoc-AAPA-Indoxyl ester [SEQ ID NO: 14]

Fmoc-AAPA-OH [SEQ ID NO: 15] (285 mg, 0.52 mmol) and CDI (102 mg, 0.63mmol) were dissolved in DCM (dry, 5 mL) in a 25 mL two-necked roundbottom flask (heated with heat gun while evacuated and cooled down underargon atmosphere) The mixture was stirred at RT under argon atmospherefor 5 min and additional 10 min while passing argon through the mixture.Then indoxyl (71 mg, 0.53 mmol) was added in one portion. Stirring whilepassing argon through the mixture was continued for 15 min, afterwardsadditional DCM (dry, 2 mL) was added and it was stirred at RT underargon atmosphere for two days. The mixture was transferred into aseparation funnel and washed with water twice. The organic phase isdried with Na₂SO₄ and concentrated to dryness. The crude product wastaken up in Et₂O (20 mL) and stirred at RT for 30 min. Then it ispurified by column chromatography on silica gel (eluent: 2% MeOH in DCM)to give 25 mg (7%) of the desired product.

ESI-MS (positive): [M+Na]⁺: 688

Example 15. Fmoc-V-Indoxyl ester

Fmoc-V-OH (501 mg, 1.48 mmol) and CDI (291 mg, 1.79 mmol) were dissolvedin DCM (dry, 6 mL). The mixture was stirred at RT under argon atmospherefor 10 min and additional 10 min while passing argon through themixture. Then additional DCM (dry, 2 ml) was added, followed by indoxyl(199 mg, 1.49 mmol) in one portion. Starring the mixture while passingargon through it was continued for 10 min, afterwards additional DCM(dry, 2 mL) was added and the mixture was stirred at RT under argonatmosphere overnight. Reaction mixture was washes with water twice,dried with Na₂SO₄, and concentrated to dryness. Crude product waspurified by column chromatography with silica gel.

Eluent: started with DCM (Column is washed with DCM under pressure untilall blue and pink color was washed down the column), then eluent waschanged to 5% MeOH in DCM.

The product came down the column with the first colored fractions.

Product was chromatographed a second time with DCM as the eluent.

Collected were fractions: 25-35 (130 mg)

-   -   36-44 (92 mg)    -   45-52 (81 mg)

Overall yield: 222 mg (33%)

Since only fractions 36-44 gave a weighable solid these fractions wereused for testing.

[M+Na]⁺: 477

Example 16: Fmoc-AAAA-Indoxyl ester [SEQ ID NO: 16]

Fmoc-AAAA-OH [SEQ ID NO: 17] (503 mg, 0.95 mmol) and CDI (191 mg, 1.18mmol) were suspended in DCM (dry, 10 mL). The mixture was stirred at RTunder argon atmosphere for 10 min and additional 10 min while passingargon through the mixture. Then indoxyl (125 mg, 0.94 mmol) was added inone portion, followed by additional DCM (dry, 5 mL). Stirring at Rtwhile passing argon through the mixture was continued for 15 min,afterwards again additional DCM (dry, 5 mL) was added, and stirringwhile passing argon through the mixture was continued for two moreminutes. Then the reaction mixture was stirred at RT under argonatmosphere overnight. Since all the solvent was evaporated overnight DCM(25 mL) was added and the mixture was stirred at RT for 5 min before itwas washed with water (2×25 mL). Organic phase was dried with Na₂SO₄ andconcentrated to dryness. Only 57 mg of crude product were obtained.Since a lot of solid was hold in the aqueous phase this solid wasfiltered off and dried. This solid was combined with the 57 mg obtainedabove. The combined solids were suspended in Et₂O (20 mL) and stirred atRT overnight. The solid was filtered off and gave 229 mg (39%) of asolid containing product and starting material (FmocA-A-A-A-OH)

[M+H]⁺: 640; [M+Na]⁺: 662

Example 17: Fmoc-APV-Indoxyl ester

Fmoc-APV-OH (482 mg, 0.92 mmol) and CDI (191 mg, 1.18 mmol) weredissolved in DCM (dry, 10 mL). The mixture was stirred at RT under argonatmosphere for 10 min and additional 10 min while passing argon throughthe mixture. Then indoxyl (125 mg, 0.94 mmol) was added in one portion,followed by additional DCM (dry, 5 mL). Stirring at RT while passingargon through the mixture was continued for 15 min, afterwards againadditional DCM (dry, 5 mL) was added, and stirring while passing argonthrough the reaction mixture was continued for 2 min and then thereaction mixture was stirred at RT under argon atmosphere overnight.Reaction mixture was washes with water (2×20 mL), organic phase wasdried with Na₂SO₄ and concentrated to dryness. The crude product wassuspended in Et₂O (20 mL) and stirred at RT for 2 h. Only little solidremained which sticked to the flask. Liquid was decanted and solidcollected (76 mg, 17%)

[M+Na]⁺: 645

Example 18: Fmoc-Phe-Indoxyl ester

Fmoc-Phe-OH (2.9 g, 7.5 mmol) and CDI (1.47 g, 9.0 mmol) are directlyweighed into a 100 mL three-necked round bottom flask. DCM (dry, 50 mL)was added and the mixture was stirred at RT for 5 min, then it wasstirred 10 min at RT while passing argon through it. Afterwardsadditional DCM (dry, 4 mL) was added, followed by indoxyl (1.00 g, 7.6mmol). Stirring at RT while passing an argon stream through the mixturewas continued for 30 min. Then additional DCM (dry, 10 mL) was added,and again the mixture was stirred at RT (with argon stream) for 20 min.Finally it is stirred at RT under argon atmosphere overnight. Reactionmixture was filled into a separation funnel, flask was washed with DCM(30 mL), and wash-DCM was filled into separation funnel as well. Water(100 mL) was added, after extraction the organic phase was collected andrefilled into a clean separation funnel. Organic phase was washed withwater (100 mL) again, dried (Na₂SO₄) and concentrated to dryness. Crudeproduct was taken up in diethyl ether (100 mL) for purification. Sinceit dissolved completely it was concentrated to dryness again andpurified by column chromatography on silica gel (eluent: DCM).Collected: fractions:

-   -   28 and 29: 152 mg    -   22-27    -   30-48

Fractions 22-27 were concentrated to dryness. Then DCM (20 mL) was addedto dissolve some of the blue colour. Solid was filtered off,re-dissolved and concentrated to dryness again to yield 132 mg of theproduct.

The filtrate was combined with fractions 30-48, yielding 1.00 g of theimpure product. Overall yield: 1.284 g (26.5%).

[M+Na]⁺: 525

Example 19: Ac-Phe-Indoxyl ester

Ac-Phe-OH (1.55 g, 7.5 mmol) and CDI are weighed directly into a 100 mLthree necked round bottom flask. DCM8dry, 50 mL) was added and themixture was stirred at RT for 5 min. Then it was stirred at RT wholepassing argon through it for 15 min. After the 15 min indoxyl was added(1.01 g, 7.6 mmol). Stirring while passing argon through the mixture wascontinued for 20 min. At that point more DCM (dry, 5 mL) was added andstirring (with argon stream) was continued for 23 min. Once more DCM(dry, 8 mL) was and stirring at RT (with argon stream) was continued for5 min. The mixture was stirred at RT under argon atmosphere overnight.Mixture was filled into separation funnel, flask was washed with DCM (20mL) which were filled into separation funnel as well. After washing withwater (80 mL) organic phase was collected and refilled into a cleanseparation funnel. Mixture was again washed with water, organic phasewas collected, dried (Na₂SO₄) and concentrated to dryness. Crude productwas purified by column chromatography on silica gel. Eluent: startedwith DCM (under pressure) until most of blue and pink color was washeddown, then solvent was changed to 2% MeOH in DCM and chromatography wascontinued without pressure. Only the almost pure fractions werecollected: 196 mg (8%).

[M+Na]⁺: 345

Example 20: Fmoc-F-V-T(Bzl)-F-

Fmoc-F-V-T(Bzl)-F-OH (1.228 mg, 1.49 mmol) was suspended in DCM (dry, 10mL). CDI (291 mg, 1.79 mmol) was added, the mixture was stirred at RTfor 10 min and additional 15 min at RT while passing argon through it.DCM (dry, 3 ml) was added and the mixture was stirred at RT (with argonstream) for two more minutes before indoxyl (200 mg, 1.50 mmol) wasadded in one portion. The mixture was stirred at RT (with argon stream)for 45 min. Afterwards additional DCM (dry, 8 mL) was added and themixture was stirred at RT for 5 more minutes before the argon line wasremoved and the mixture was stirred at RT under argon atmosphereovernight. DCM (20 mL) was added, the remaining blue solid was filteredoff and washed with DCM (20 mL). The filtrate was filled into aseparation funnel and washed with water twice. The organic phase wasdried (Na₂SO₄) and concentrated to dryness. The crude product was takenup in DCM (7 mL) and diethyl ether was added (20 mL). The mixture wasstirred for 2 h at RT, then the precipitate (58 mg, 5%) was filtered offand washed with diethyl ether (20 mL).

[M+Na]⁺: 962

Example 21: β-Lactam indoxyl ether

Indoxyl (327 mg, 2.46 mmol) was weighed into a 25 mL three-necked roundbottom flask (flask was heated in vacuo with heat gun and cooled downunder argon atmosphere). THF (dry, 6 mL) was added and the mixture wasstirred at RT while streaming argon through the mixture for 10 min. Thenpotassium tert-butoxide (228 mg, 2.03 mmol) was added in one portion andstirring at RT with a permanent argon stream was continued for 15 min.At this point 4-methoxybenzyl3-Chloromethyl-7-(2-phenylacetamido)-3-cephem-4-carboxylate (1.01 g,2.05 mmol) was added in one portion. Stirring the mixture at RT with asteady argon stream was continued for additional 30 min. The product wasdetected via ESI-MS. ESI-MS (positive): [M+Na]+=606; [M+K]+=622.

Example 22. N-(Propargyl)-(RBB)

Class: Dye conjugate based on Remazol Brilliant Blue R, 1,3-dipolar[3+2] cycloaddition (“click chemistry”)

Synthesis: Reaction of Remazol Brilliant Blue R with propargylaminehydrochloride was performed under normal conditions in saturated aq.NaHCO₃. Reaction monitoring was done with ESI-MS (-p). The product wasextracted with ethyl acetate from the aqueous phase and precipitatedwith adequate purity for use in the subsequent reaction (1,3-dipolar[2+3]-cycloaddition). ESI-MS (negative) [M−H]⁻: 538.

Example 23. Fmoc Protected Reagent(Fmoc-V-{2-hydroxy-3-[1-(5-(N-RBB-methyl)1,2,3-triazolyl)}propylamide)(Fmoc-V-triazol-RBB)

Example 22 was reacted with FMOC-valine-N-(2-hydroxy-3-azidopropyl,amide using click chemistry conditions (Hünig's base, copper catalysis).The reaction product is used in further examples below.

Example 24.H-V-{2-hydroxy-3-[1-(5-(N-RBB-methyl)1,2,3-triazolyl)}propylamide(H-V-triazol-RBB)

Class: Dye conjugate based on Remazol Brilliant Blue R, amino acidbuilding block for peptide synthesis.

Synthesis: Reaction of Fmoc protected reagent (Fmoc-V-Triazole-RBB,Example 23) with piperidine in methanol was performed under normalconditions. Reaction monitoring was done with ESI-MS (-p). The productprecipitated after drying in vacuo with adequate purity for use in thesubsequent reaction (amidation). Yield: quantitative (crude product).ESI-MS (negative) [M−H]⁻: 753.

Example 25.Fmoc-AAPVAV-{2-hydroxy-3-[1-(5N-RBB-methyl)1,2,3-triazolyl)}propylamide(Fmoc-AAPVAV-triazol-RBB) [SEQ ID NO: 18]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: A stirred mixture of pentapeptide Fmoc-AAPVA-OH [SEQ ID NO:19] (1 equiv) and HOB^(t)·H₂O (1.2 equiv) in DCM was treated with DCC(1.2 equiv) at ambient temperature for 15 min.H-V-{2-hydroxy-3-[1-(5-(N-RBB-methylo)1,2,3-triazolyl)}propylamide (1equiv; Example 24) was added in one portion and the reaction mixture wasstirred at room temperature until reaction monitoring via ESI-MS (-p)indicated consumption of starting materials. Simple removal of volatilesin vacuo yielded a crude product as blue amorphous solid. Yield: ˜83%(crude product). ESI-MS (negative) [M−H]⁻: 1385.

Example 26.Fmoc-AAPV-{2-hydroxy-3-[1-(5-(N-RBB-methyl)1,2,3-triazolyl)}propylamide(Fmoc-AAPV-triazol-RBB) [SEQ ID NO: 2]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: A stirred mixture of tripeptide Fmoc-AAP-OH (1 equiv) andHOBt·H₂O (1.2 equiv) in DCM was treated with DCC (1.2 equiv) at ambienttemperature for 15 min.H-V-{2-hydroxy-3-[1-(5-(N-RBB-methylo)1,2,3-triazolyl)}propylamide (1equiv; Example 24) was added in one portion and the reaction mixture wasstirred at room temperature until reaction monitoring via ESI-MS (-p)indicated consumption of starting materials. Aqueous work-up yielded acrude product as blue amorphous solid. Yield: ˜90% (crude product).ESI-MS (negative) [M−H]⁻: 1214.

Example 27.H-AAPVAV-{2-hydroxy-3-[1-(5-(N-RBB-methyl)1,2,3-triazolyl)}propylamide(H-AAPVAV-triazol-RBB) [SEQ ID NO: 20]

Dye-peptide conjugate based on Remazol Brilliant Blue R. Synthesis:Fmoc-AAPVAV-{2-hydroxy-3-[1-(5-(N-RBB-methylo)1,2,3-triazolyl)}propylamide[SEQ ID NO: 18] (1 equiv; Example 25) was treated with a mixture ofmethanol/piperidine (2:1) at ambient temperature for 45 min whilestirring. Reaction monitoring via ESI-MS (-p) indicated consumption ofstarting materials. Simple evaporation of volatiles in vacuo yielded acrude product as blue amorphous solid. Yield: quantitative (crudeproduct). ESI-MS (negative) [M−H]⁻: 1162.

Example 28.H-AAPV-{2-hydroxy-3-[1-(5-(N-RBB-methyl)1,2,3-triazolyl)}propylamide)(H-AAPV-triazol-RBB) [SEQ ID NO: 21]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis:Fmoc-AAPV-{2-hydroxy-3-[1-(5-(N-RBB-methylo)1,2,3-triazolyl)}propylamide(1 equiv [SEQ ID NO: 2]; Example 26) was treated with a mixture ofmethanol/piperidine (2:1) at ambient temperature for 45 min whilestirring. Reaction monitoring via ESI-MS (-p) indicated consumption ofstarting materials. Simple evaporation of volatiles in vacuo yielded acrude product as blue amorphous solid. Yield: quantitative (crudeproduct). ESI-MS (negative) [M−H]⁻: 992.

Example 29. RB-Amine (H-RBB)

Class: Dye conjugate based on Remazol Brilliant Blue R, Startingmaterial for Amination/Amidation

Synthesis: Reaction of Remazol Brilliant Blue R with ammonium hydroxidesolution was performed under normal conditions, with initial cooling inan ice bath. Reaction was monitored via ESI-MS (-p). The product wasfiltered from the aqueous phase and precipitated with adequate purityfor use in the subsequent reaction (amidation). Yield: >64% (crudeproduct). ESI-MS (negative) [M−H]⁻: 500.

Example 30 (RBB)-amido-Fmoc-Alanine (Fmoc-A-RBB)

Class: Dye conjugate based on Remazol Brilliant Blue R, amino acidbuilding block for peptide synthesis.

Synthesis: Reaction of H-RBB (Example 29) with Fmoc-alaninepentafluorophenyl ester was performed under normal conditions inethanol. Reaction monitoring was done with ESI-MS (-p). The product wasextracted from the aqueous phase with dichloromethane (DCM) andprecipitated after drying in vacuo with adequate purity for use in thesubsequent reaction (deprotection/amidation). Yield: >90% (crudeproduct). ESI-MS (negative) [M−H]⁻: 793.

Example 31. RBB-amido-Alanine-NH₂ (H-A-RBB)

Class: Dye conjugate based on Remazol Brilliant Blue R, amino acidbuilding block peptide synthesis.

Synthesis: Reaction of Fmoc-A-RBB (Example 30) with piperidine inmethanol was performed under normal conditions. Reaction monitoring wasdone with ESI-MS (-p). The product precipitated after drying in vacuowith adequate purity for use in the subsequent reaction (amidation).Yield: quantitative (crude product). ESI-MS (negative) [M−H]⁻: 571.

Example 32. Fmoc-AAPVAA-RBB [SEQ ID NO: 22]

Class: Dye-peptide conjugate conjugate based on Remazol Brilliant Blue R

Synthesis: A stirred mixture of pentapeptide Fmoc-AAPVA-OH [SEQ ID NO:19] (1 equiv) and HOBt·H₂O (1.2 equiv) in DCM was treated with DCC (1.2equiv) at ambient temperature for 15 min. H-A-RBB (1 equiv, Example 31)was added in one portion and the reaction mixture was stirred at roomtemperature until reaction monitoring via ESI-MS (-p) indicatedconsumption of starting materials. Aqueous work-up yielded a crudeproduct as blue amorphous solid. Yield: −58% (crude product). ESI-MS(negative) [M−H]⁻: 1202.

Example 33. H-AAPVAA-RBB [SEQ ID NO: 23]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: Fmoc-AAPVAA-RBB [SEQ ID NO: 22] (1 equiv, Example 32) wastreated with a mixture of methanol/piperidine (2:1) at ambienttemperature for 45 min while stirring. Reaction monitoring via ESI-MS(-p) indicated consumption of starting materials. Simple evaporation ofvolatiles in vacuo yielded a crude product as blue amorphous solid.Yield: quantitative (crude product). ESI-MS (negative) [M−H]⁻: 980.

Example 34. Acrylamido-AAPVAA-RBB [SEQ ID NO: 22]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: H-AAPVAA-RBB [SEQ ID NO: 23] (1 equiv; Example 33) wasdissolved in methanol and treated with acrylic acid pentafluorophenylester (1.2 equiv) at ambient temperature while stirring. Reactionmonitoring via ESI-MS (-p) indicated consumption of starting materialsafter ˜12 h (overnight). Simple evaporation of volatiles in vacuoyielded crude product as blue amorphous solid adequately pure for thenext step. Yield: >95% (crude product). ESI-MS (negative) [M−H]⁻: 599.

Example 35. Fmoc-VA-RBB

Class: Dye conjugate based on Remazol Brilliant Blue R, amino acidbuilding block for peptide synthesis.

Synthesis: A solution of H-A-RBB (1 equiv; Example 31) in methanol wastreated with Fmoc-Valine-OPfp ester at ambient temperature whilestirring. After reaction monitoring via ESI-MS (-p) indicatedconsumption of starting materials, the reaction mixture was subjected toaqueous work-up and extracted with DCM. Evaporation of DCM in vacuoyielded a crude product as blue amorphous solid. Yield: −48% (crudeproduct). ESI-MS (negative) [M−H]⁻: 892.

Example 36. H-VA-RBB

Class: Dye conjugate based on Remazol Brilliant Blue R, amino acidbuilding block for peptide synthesis;

Synthesis: Fmoc-VA-RBB (1 equiv; Example 35) was treated with a mixtureof methanol/piperidine (2:1) at ambient temperature for 45 min whilestirring. Reaction monitoring via ESI-MS (-p) indicated consumption ofstarting materials. Simple evaporation of volatiles in vacuo yielded acrude product as blue amorphous solid. Yield: quantitative (crudeproduct). ESI-MS (negative) [M−H]⁻: 670.

Example 37. Fmoc-AAPFA-RBB [SEQ ID NO: 24]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: A stirred mixture of tetrapeptide Fmoc-AAPF-OH [SEQ ID NO:13] (1 equiv) and HOBt·H₂O (1 equiv) in DCM was treated with DCC (1equiv) at ambient temperature for 15 min. H-A-RBB (0.8 equiv; Example31) was added in one portion and the reaction mixture was stirred atroom temperature until reaction monitoring via ESI-MS (-p) indicatedconsumption of starting materials. Aqueous work-up and evaporation ofvolatiles in vacuo yielded a crude product as blue amorphous solid.Yield: −98% (crude product). ESI-MS (negative) [M−H]⁻: 1179.

Example 38. H-AAPFA-RBB [SEQ ID NO: 25]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: Fmoc-AAPFA-RBB [SEQ ID NO: 24] (1 equiv) (example 37) wastreated with a mixture of methanol/piperidine (2:1) at ambienttemperature for 45 min while stirring. Reaction monitoring via ESI-MS(-p) indicated consumption of starting materials. Simple evaporation ofvolatiles in vacuo yielded a crude product as blue amorphous solid.Yield: quantitative (crude product). ESI-MS (negative) [M−H]⁻: 957.

Example 39. Fmoc-AAPVA-RBB [SEQ ID NO: 26]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: A stirred mixture of pentapeptide Fmoc-AAPVA-OH [SEQ ID NO:27] (1 equiv) and HOBt·H₂O (1.2 equiv) in DCM was treated with DCC (1.2equiv) at ambient temperature for 15 min. H-RBB (1 equiv, Example 29)was added in one portion and the mixture was stirred at room temperatureuntil reaction monitoring via ESI-MS (-p) indicated consumption ofstarting materials. Aqueous work-up yielded a crude product as blueamorphous solid. Yield: −23% (crude product). ESI-MS (negative) [M−H]⁻:1131.

Alternate Synthesis 1: A stirred mixture of tripeptide Fmoc-AAP-OH (1equiv) and HOBt·H₂O (1 equiv) in DCM was treated with DCC (1 equiv) atambient temperature for 15 min. H-VA-RBB (0.8 equiv; Example 36) wasadded in one portion and the reaction mixture was stirred at roomtemperature until reaction monitoring via ESI-MS (-p) indicatedconsumption of starting materials (−15 h, overnight). Aqueous work-upand evaporation of volatiles in vacuo yielded adequately pure crudeproduct as blue amorphous solid. Yield: −70% (crude product). ESI-MS(negative) [M−H]⁻: 1131.

Alternate synthesis 2: Fmoc-AAPVA-OH [SEQ ID NO: 27] (110 mg) and HOBt(50 mg) were suspended in DMF (10 mL). DCC (50 mg) was added. Afterstirring for 5 min, H-RBB (100 mg; Example 29) dissolved in DMF (3 mL)was added. After stirring for 2 h, the reaction was complete asindicated by MS. The crude mixture was taken forward for Fmocdeprotection (see Alternate Synthesis in Example 40). ESI-MS (negative)[M−H]⁻: 1131.

Example 40. H-AAPVA-RBB [SEQ ID NO: 28]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: Fmoc-AAPVA-RBB [SEQ ID NO: 26] (1 equiv; Example 39) wastreated with a mixture of methanol/piperidine (2:1) at ambienttemperature for 45 min while stirring. Reaction monitoring via ESI-MS(-p) indicated consumption of starting materials. Simple evaporation ofvolatiles in vacuo yielded a crude product as blue amorphous solid.Yield: quantitative (crude product). ESI-MS (negative) [M−H]⁻: 909.

Alternate synthesis: Using the crude DMF mixture of Fmoc-AAPVA-RBB [SEQID NO: 26](from Alternate Synthesis 2 in Example 39), piperidine (200μL) was added and the mixture was stirred overnight. The solvent wasevaporated, and the residue was re-dissolved in methanol (˜50 mL). Afterconcentration to 10 ml, the deep blue supernatant was poured intodiethyl ether (200 mL). The precipitate was isolated and dried to yield145 mg of a fine blue powder (H-AAPVA-RBB) [SEQ ID NO: 28]. ESI-MS(negative) [M−H]⁻: 909.

Example 41. Fmoc-V-RBB

Class: Dye conjugate based on Remazol Brilliant Blue R, amino acidbuilding block for peptide synthesis.

Synthesis: A solution of H-RBB (1 equiv; Example 29) in methanol wastreated with Fmoc-Valine-OPfp ester at ambient temperature whilestirring. After reaction monitoring via ESI-MS (-p) indicatedconsumption of starting materials, the reaction mixture was subjected toaqueous work-up and extracted with DCM. Evaporation of DCM in vacuoyielded a crude product as white amorphous solid. Yield: ˜31% (crudeproduct). ESI-MS (negative) [M−H]⁻: 821.

Example 42. H-V-RBB

Class: Dye conjugate based on Remazol Brilliant Blue R, amino acidbuilding block for peptide synthesis.

Synthesis: Fmoc-V-RBB (1 equiv; Example 41) was treated with a mixtureof methanol/piperidine (2:1) at ambient temperature for 45 min whilestirring. Reaction monitoring via ESI-MS (-p) indicated consumption ofstarting materials. Simple evaporation of volatiles in vacuo yielded acrude product as blue amorphous solid. Yield: quantitative (crudeproduct). ESI-MS (negative) [M−H]⁻: 599.

Example 43. Fmoc-AAPV-RBB [SEQ ID NO: 2]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: A stirred mixture of tripeptide Fmoc-AAP-OH (1 equiv) andHOBt·H₂O (1 equiv) in DCM was treated with DCC (1 equiv) at ambienttemperature for 15 min. H-V-RBB (0.8 equiv; Example 42) was added in oneportion and the reaction mixture was stirred at room temperature untilreaction monitoring via ESI-MS (-p) indicated consumption of startingmaterials (˜15 h, overnight). Aqueous work-up and evaporation ofvolatiles in vacuo yielded adequately pure crude product as blueamorphous solid. Yield: ˜70% (crude product). ESI-MS (negative) [M−H]⁻:1060.

Example 44. H-AAPV-RBB [SEQ ID NO: 29]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: Fmoc-AAPV-RBB [SEQ ID NO: 2] (1 equiv; Example 43) wastreated with a mixture of methanol/piperidine (2:1) at ambienttemperature for 45 min while stirring. Reaction monitoring via ESI-MS(-p) indicated consumption of starting materials. Simple evaporation ofvolatiles in vacuo yielded adequately pure crude product as blueamorphous solid. Yield: quantitative (crude product). ESI-MS (negative)[M−H]⁻: 838.

Example 45. Fmoc-AAPF-RBB [SEQ ID NO: 12]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: A stirred mixture of tetrapeptide Fmoc-AAPF-OH [SEQ ID NO: 4](1 equiv) and HOBt·H₂O (1 equiv) in DCM were treated with DCC (1 equiv)at ambient temperature for 15 min. H-RBB (0.8 equiv; Example 29) wasadded in one portion and the reaction mixture was stirred at roomtemperature until reaction monitoring via ESI-MS (-p) indicatedconsumption of starting materials. Aqueous work-up and evaporation ofvolatiles in vacuo yielded a crude product as blue amorphous solid.Yield: ˜98% (crude product). ESI-MS (negative) [M−H]⁻: 1108.

Example 46. H-AAPF-RBB [SEQ ID NO: 30]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: Fmoc-AAPF-RBB [SEQ ID NO: 12] (1 equiv) (example 45) wastreated with a mixture of methanol/piperidine (2:1) at ambienttemperature for 45 min while stirring. Reaction monitoring via ESI-MS(-p) indicated consumption of starting materials. Simple evaporation ofvolatiles in vacuo yielded a crude product as blue amorphous solid.Yield: quantitative (crude product). ESI-MS (negative) [M−H]⁻: 886.

Example 47. Acrylamido-AAPF-RBB [SEQ ID NO: 12]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: H-AAPF-RBB (1 equiv; Example 46) was dissolved in methanoland treated with acrylic acid pentafluorophenyl ester (3 equiv) atambient temperature while stirring. Reaction monitoring via ESI-MS (-p)indicated consumption of starting materials after ˜12 h (overnight).Simple evaporation of volatiles in vacuo yielded crude product which wasagain dissolved in DCM leaving insoluble white byproducts behind.Subsequent evaporation in vacuo upon filtration yielded the product asblue amorphous solid adequately pure for the next step. Yield: ˜98%(crude product). ESI-MS (negative) [M−H]⁻: 94

Example 48. Fm-Valin-(3-Azido-2-hydroxy)-propylamide

Class: Amino acid building block for Peptide synthesis, substrate for1,3-dipolar [3+2]-cycloaddition.

Synthesis: Fmoc-N-(2-epoxypropyl-1-amido)-valine (1 equiv) was dissolvedin DCM. Sodium azide (excess) was dissolved in water and the aqueousphase was added to the organic solution at ambient temperature whilestirring (>1000 rpm). Catalytic amount of phase transfer catalysttetra-n-butyl ammonium hydrogen sulfate was added followed by subsequentadditions of catalytic amounts of sulfuric acid. The latter was consumedby time and needed to be refreshed on a regular basis until reactionmonitoring via ESI-MS (+p) indicated full conversion of startingmaterials. Aqueous work-up and evaporation of volatiles in vacuo yieldedcrude colorless to beige product as amorphous solid. Yield: 66% (crudeproduct). ESI-MS (positive) [M+Na]⁺: 460.

Example 49. Acrylamido-AAPVA-RBB [SEQ ID NO: 26]

Class: Dye-peptide conjugate based on Remazol Brilliant Blue R.

Synthesis: H-AAPVA-RBB [SEQ ID NO: 28] (1 equiv; Example 40) wasdissolved in methanol and treated with acrylic acid pentafluorophenylactive ester (1.2 equiv) at ambient temperature while stirring. Reactionmonitoring via ESI-MS (-p) indicated consumption of starting materialsafter ˜12 h (overnight). Simple evaporation of volatiles in vacuoyielded crude product as blue amorphous solid adequately pure for thenext step. Yield: >95% (crude product). ESI-MS (negative) [M−H]⁻: 963.

Example 50. Bis-Aminoethyl-(Remazol Black B)

Class: Dye conjugate based on Remazol Black B, Amination/Amidationsubstrate

Synthesis: Remazol Black B (1 equiv) was dissolved in water and treatedwith aqueous ammonia (28-30%, excess) at ambient temperature whilestirring. Reaction monitoring via ESI-MS (-p) indicated full conversionof starting materials after ˜20 h (overnight). Simple evaporation ofvolatiles in vacuo yielded crude product as black amorphous solid withslight violet gloss adequately pure for the next step. Yield:quantitative (crude product). ESI-MS (negative) [M−H]⁻: 740.

Example 51. Aminoethyl-Remazol Brilliant Violet

Class: Azo-Dye conjugate based on Remazol Brilliant Violet 5R,Amination/Amidation substrate.

Synthesis: Remazol Brilliant Violet 5R (1 equiv) was dissolved in waterand treated with aqueous ammonia (28-30%, excess) at ambient temperaturewhile stirring. Reaction monitoring via ESI-MS (-p) was not possible dueto the fact that neither starting materials nor products were detectablein ESI-MS ionization. After prolonged period of reaction time (9 d) toensure full conversion simple evaporation of volatiles in vacuo yieldedcrude product as dark violet amorphous solid. Yield: ˜82% (crudeproduct).

Example 52. Fmoc-AAPF-aminoethyl-(Remazol Black B) [SEQ ID NO: 12]

Class: Dye conjugate based on Remazol Black B, Tetrapeptide buildingblock, Cathepsin substrate.

Synthesis: A stirred mixture of tetrapeptide Fmoc-AAPF-OH [SEQ ID NO:13] (1 equiv) and HOBt·H₂O (1 equiv) in DCM was treated with DCC (1equiv) at ambient temperature for 15 min. Bis-Aminoethyl-(Remazol BlackB) (0.8 equiv; Example 50) was added in one portion and the reactionmixture was stirred at room temperature until reaction monitoring viaESI-MS (-p) indicated consumption of starting materials. Used in nextstep without further purification.

Example 53. Fmoc-AAPF-aminoethyl-(Remazol Black B)-(N-acetyl)-ethylamide[SEQ ID NO: 12]

Class: Dye-peptide conjugate based on Remazol Black B, Cathepsinsubstrate.

Synthesis: The stirred reaction mixture forFmoc-AAPF-aminoethyl-(Remazol Black B) [SEQ ID NO: 12] (Example 52) wastreated with excess acetic anhydride at ambient temperature for 4 h.When reaction monitoring by ESI-MS (-p/+p) indicated consumption ofstarting materials, the reaction mixture was filtered and the filtratewas evaporated in vacuo yielding black amorphous powder as a crudeproduct. The crude product was subsequently used without additionalpurification. Identity was imputed through reaction with elastase toyield the mono-acetylated-Remazol Black B-amino derivative.

Example 54. CMC 9M31F-AAPFA-RBB [SEQ ID NO: 24]

Class: Full prototype assembly (Back bone/cutting unit/Dye), Cathepsinsubstrate.

Synthesis: Blanose CMC 9M31F (1 equiv) was dissolved in water and wastreated with HOBt (1.2 equiv) followed by EDC·HC1 (1.2 equiv) for 0.5 hwhile stirring at ambient temperature. H-AAPFA-RBB [SEQ ID NO: 25] (0.1equiv; Example 38) was added and stirring was continued overnight (>14h). The following day, the reaction mixture was purified by dialysisyielding a blue jelly as crude product. Yield: depends on degree ofsubstitution; 1 g Blanose provides ca. 1 g product. The resultingproduct was a polymer and was not easily characterised by spectroscopy.It was, however, blue, and the blue color is associated with the high MWfraction following dialysis in a 10 kDa membrane. Reaction with elastasewas detected using ESI MS to detect the release of the dye.

Example 55. CMC 9M31F-AAPF-RBB [SEQ ID NO: 12]

Class: Full prototype assembly (Back bone/cutting unit/Dye), Cathepsinsubstrate.

Synthesis: Blanose CMC 9M31F (1 equiv) was dissolved in water and wastreated with HOBt (1.2 equiv) followed by EDC·HC1 (1.2 equiv) for 0.5 hwhile stirring at ambient temperature. H-AAPF-RBB [SEQ ID NO: 30] (0.1equiv; Example 46) was added and stirring was continued overnight (>14h). The following day, the reaction mixture was purified by dialysisyielding a blue jelly as crude product. Yield: depends on degree ofsubstitution; 1 g Blanose provides ca. 1 g product. The resultingproduct was a polymer and was not easily characterised by spectroscopy.It was, however, blue, and the blue color is associated with the high MWfraction following dialysis in a 10 kDa membrane. Reaction with elastaseis detected using ESI MS to detect the release of the dye.

Example 56. AAPVA-RBB Bound to Paper Membrane [SEQ ID NO: 26]

Class: Full prototype assembly on a surface or solid phase “6651”(anchor region/enzyme-labile or enzyme-reactive region/indicatorregion), Elastase substrate.

Synthesis: Paper membrane (1 equiv) was soaked in DMF and subsequentlytreated with HOBt-H₂O (˜1.2 equiv), DCC (˜1.2 equiv) and H-AAPVA-RBB[SEQ ID NO: 28] (˜1 equiv; Example 40) at ambient temperature whilestirring. After two days of reaction time, paper membranes were filteredoff, washed with water, saturated aq. NaHCO₃, ethanol, ethyl acetate,and dried with diethyl ether. After treatment, the membranes kept aslight greenish color, indicating a very low degree of loading. Yield:n.a. The resulting product was a polymer and was not easilycharacterised by spectroscopy.

Example 57. Fmoc-M-RBB

Class: Methionine-Dye conjugate based on Remazol Brilliant Blue, Eductfor Amination/Amidation substrate.

Synthesis: H-RBB (1 equiv; Example 29) was dissolved in methanol andFmoc-methionine pentafluorophenyl ester (3 equiv) was added at ambienttemperature while stirring. Reaction monitoring via ESI-MS (-p)indicated consumption of starting materials and the reaction was stoppedby evaporation of any volatiles in vacuo. A blue amorphous solid wasobtained as crude product, sufficiently pure for the next step. Yield:quantitative (based on Dye). ESI-MS (negative) [M−H]⁻: 853.

Example 58. H-M-RBB

Class: Methionine-Dye conjugate based on Remazol Brilliant Blue,Amination/Amidation substrate.

Synthesis: Fmoc-M-RBB (1 equiv; Example 57) was dissolved in a mixtureof methanol and piperidine (2:1 v/v) at ambient temperature whilestirring. After a short time (<1 h), reaction monitoring via ESI-MS (-p)indicated consumption of starting materials, and the reaction wasstopped by evaporation of any volatiles in vacuo. A blue amorphous solidremained as crude product which was taken up in acetonitrile. Insolublewhite byproducts were filtered off and again volatiles were removed invacuo. The blue amorphous product obtained was sufficiently pure for thenext step. Yield: quantitative. ESI-MS (positive) [M+H]⁺: 633.

Example 59. Fmoc-AAAPM-RBB [SEQ ID NO: 31]

Class: Dye conjugate based on Remazol Brilliant Blue, Pentapeptidebuilding block, Cathepsin substrate.

Synthesis: A stirred mixture of tetrapeptide Fmoc-AAAP-OH [SEQ ID NO:32] (1 equiv) and HOBt·H₂O (1.2 equiv) in DCM was treated with DCC (1.2equiv) at ambient temperature for 15 min. H-M-RBB (1 equiv; Example 58)was added in one portion and the reaction mixture was stirred at roomtemperature until reaction monitoring via ESI-MS (-p) indicatedconsumption of starting materials. Reaction mixture was further dilutedwith DCM, urea species filtered off, and the filtrate was subjected toaqueous work-up. After drying over sodium sulfate evaporation ofvolatiles in vacuo yielded blue amorphous solid as crude productsufficiently pure for the next step. Yield: >85%. ESI-MS (negative)[M−H]⁻: 1163.

Example 60. H-AAAPM-RBB [SEQ ID NO: 33]

Class: Dye conjugate based on Remazol Brilliant Blue, Pentapeptidebuilding block, Cathepsin substrate.

Synthesis: Fmoc-AAAPM-RBB [SEQ ID NO: 31] (1 equiv; Example 59) wasdissolved in a mixture of methanol and piperidine (2:1 v/v) at ambienttemperature while stirring. After a short time (<1 h), reactionmonitoring via ESI-MS (-p) indicated consumption of starting materials,and the reaction was stopped by evaporation of any volatiles in vacuo. Ablue amorphous solid remained as crude product which was taken up inacetonitrile. Insoluble white byproducts were filtered off and againvolatiles were removed in vacuo. A blue amorphous product was obtained,sufficiently pure for further investigations. Yield: quantitative.ESI-MS (negative) [M−H]⁻: 941.

Example 61. Poly-N-acroyl-S-tritylcysteine

To a solution of poly-N-nitrophenyl acrylate (PAA) (722 mg) in dry DMF(6 mL) was added H-Cys(Trt)-OH (1030 mg) and triethylamine (450 μL). Thereaction mixture was carefully moved {i.e., rolled), with occasionalultrasonication, until all solid was dissolved. Then the mixture waswarmed to 54° C. for 1 5h with occasional shaking. When reactionmonitoring by MS indicated disappearance of H-Cyst(Trt)-OH, ethanolamine(250 μL.) was added to quench unreacted sites and warming was continuedfor an additional 30 min. The mixture was poured into methanol (80 mL).All precipitates were isolated, re-dissolved with DMF (5 mL) andre-precipitated with methanol (100 mL) by addition of formic acid (100μL) or saturated aq. calcium chloride solution (100 μL). The precipitatewas isolated, stirred for 2 h with methanol (100 mL) and filtered. Afterwashing with methanol and diethyl ether, 540 mg of the product wasisolated.

Combination of the supernatants, addition of formic acid until the colorof nitrophenol disappeared and re-precipitation of the precipitate asdescribed above yielded an additional 405 mg of product. Total yield:approx. 70%. (Due to the polymeric nature of the material, no specificcharacterization was possible. However, the disappearance oftrityl-cysteine is a strong support for the proposed structure.)

Using this procedure, other amines were loaded to the activatedpolyacrylate (see Table 1). When performed on a small scale, gelchromatography of the crude reaction mixture in DMF proved to be asuitable purification method. (Due to the polymeric nature of thematerials, no specific characterization was possible. However, materialpassed through Sephadex is of high molecular weight and blue, thusproving attachment of the dye to the polymer).

TABLE 1 PAA amine for Purification Entry (mg) amine quench method 1 579H-AAPVA-CV, ethanolamine chromatography SEQ ID NO: 28 over Sephadex (asmixture in DMF, 15 mg) LH 20 ® (example 71) 2 500 tris- di-n-Precipitation hydroxymethylmethylamine octylamine (“TRIS”) (60 mg) 3 450S-Tritylcysteine (240 mg) butlyamine Precipitation 4 620S-Tritylcysteine (52 mg)  N_(a)-Boc- Precipitation Lysine

Example 62. Linking of a peptide-dye conjugate to a polymer(poly-(N-acroyl-Boc-Lys-OH)-co-(acroyl-Cys(Trt)-OH)

H-AAPVA-RBB [SEQ ID NO: 28] (52 mg; Example 40) was dissolved in DMF (1mL) and combined with ethyl diisopropylamine (10 μL,). Chloroacetic acidanhydride (10.8 mg) was added. The mixture was stirred for 1 h, thenadditional chloroacetic acid anhydride (8.5 mg) was added. Afterstirring for further 2 h, MS indicated predominant conversion. 500 μL ofthis solution was combined withpoly-(N-acroyl-Boc-Lys-OH)-co-(acroyl-Cys(Trt)-OH) (104 mg; entry 4 inTable 1) and ethyl diisopropylamine (70 μL). The mixture was heated to60° C. overnight, then to 80° C. for 40 min. The reaction mixture wasapplied to a Sephadex LH 20® column for purification; the front-elutingblue zone was isolated to yield 85 mg of product. (Due to the polymericnature of the materials, no specific characterization was possible.However, material passed through Sephadex is of high molecular weightand blue, thus proving attachment of the dye to the polymer). Treatmentwith elastase released Ala-RBB, as found by MS-analysis (m/z=571,negative mode).

The product of Step A was dissolved with DCM (1 mL) and methanol (0.5mL) and treated for 40 min with trifluoroacetic acid (0.5 mL). Allvolatiles were removed in vacuo and the product was precipitated byaddition of diethylether (100 mL). The product is a blue polymericsolid, with no observable mass in the negative mode below m/z=1200.Treatment with elastase released A-RBB, as found by MS-analysis(m/z=571, negative mode).

Step C:

Poly-N-acroylcystein and 530 mg of Poly-N-acroyl-S-tritylcystein weredissolved with a mixture of 18 mL of dichloromethane, 1 mL oftriisopropylsilane, and 2 mL of trifluoroacetic acid. 0.5 mL of waterwas added and the mixture was stirred overnight. The mixture wasevaporated and washed with water and diethylether. (Due to the polymericnature of the materials, no specific characterization was possible).

Example 63. Exemplary Construction of a High-Load Amino-Peptide-DyeConjugate Linked to a Polymer

a) H-AAPVA-RBB [SEQ ID NO: 28] (68 mg; Example 40) was dissolved withDMF (1 mL). Chloroacetic anhydride (16 mg) was added followed by ethyldiisopropylamine (100 μL). After stirring for 1 h, Jeffamine® 900 (80mg) was added, and the mixture was warmed to 70° C. for 4 h.Poly-p-nitrophenylacrylate (75 mg) and diisopropylamine (100 μL) wereadded. After stirring for 2 h, residual reactive sites of the polymerwere quenched with n-butylamine. (150 μL) The blue polymeric product wasisolated by gel chromatography over Sephadex LH 20® to yield 105 mg.(Due to the polymeric nature of the materials, no specificcharacterization was possible. However, the material passed throughSephadex is of high molecular weight and blue, thus proving attachmentof the dye to the polymer).

b) 500 μL, of a 58 mM solution of Chloroacetamido-AAPVA-RBB [SEQ ID NO:31] (prepared in Step A) and 4 μL of ethylenediamine are combined,heated to 60° C. overnight and to 80° C. for 1h. 72 mg ofpoly-(4-nitrophenyl)acrylate are dissolved in 500 μL of DMF and 70 μL oftriethylamine are added and the mixture is stirred overnight. 300 μL ofa 1M aq. solution of KOH are added, and the mixture is rolled foradditional 45 min. A precipitate was isolated by centrifugation,subsequently dissolved with 0.1M KOH and precipitated with formic acid.The precipitate was dissolved with 1 ml of saturated aq. NaHCO₃andpassed through a column of 6 g of Sephadex LH20. Drying in vacuo yields84 mg of a bluish solid.

c) R=H, CH₂C(═O)-AAAPV-RBB [SEQ ID NO: 34]. 190 mg of H-AAAPV-RBB [SEQID NO: 34] (example 68c) are dissolved in 3 mL of DMF and 40 mg ofchloroacetic anhydride are added. 40 μL of diisopropyl ethyl amine areadded, and the mixture was stirred for 15 min at RT. A 2^(nd) batch ofchloroacetic anhydride (17 mg) and of diisopropyl ethylamine (20 μL),each, was added, and stirring is continued for 1 h. The reaction mixturewas applied to silica gel and chromatographed with a gradient ofcyclohexane-methanol-dichloromethan from 16-3-3 to 3-1-1, containing0.2% of formic acid. The product fraction contained 69 mg, which aredissolved with 1 mL of DMF. 20 μL of diisopropyl ethyl amine are added,then 12 μL of Jeffamine EDR 176. The reaction mixture was agitated for72h at RT. Analysis with a mass spectrometer reveals a mixture ofalkylation products with the peptide being added 1, 2, or 3 times to theamine (negative mode, m/z=1125=monoalkyl, single charge; m/z=1038=doublealkyl, double charge; m/z=1513=triple alkyl, double charge; andm/z=1009=triple alkyl, triple charge). The reaction mixture was suppliedwith 38 mg of poly-p-nitrophenylacrylate, agitated for Ih at RT and thenkept 90 min at 54° C. 250 μL of N,N-dioctyl amine are added, and themixture is kept at 54° C. for further 3h with occasional shaking. Theproduct was isolated by precipitation with acetone and chromatography ofthe precipitate after solution in methanol through a column of 6 g ofSephadex LH 20 with water-methanol 3-1 (highest molecular weightfraction is collected) to yield 31 mg of a water soluble material. Thesame procedure can be applied to H-AAAPVV-RBB [SEQ ID NO: 35] (example68c).

Example 64. Fmoc-AAAPV-(diaminobenzoic acid) [SEQ ID NO: 36]

Fmoc-AAAPV-OH [SEQ ID NO: 11] (710 mg) was dissolved with DMF (5 mL).HOBt (210 mg) and DCC (240 mg) were added subsequently, and the mixturewas stirred for 15 min at RT. 3,4-Diaminobenzoic acid (160 mg) andpyridine (100 μL) were dissolved with DMF (1 mL) and added to reactionmixture with stirring. After 14 h, the solvent was evaporated, and theresidue was purified by silica gel chromatography withcyclohexane/DCM/methanol (4/2/1), containing 0.5% of formic acid, toyield 400 mg of product. ESI-MS (positive) [M+H]=784.

Example 65. H-AAAPV-(diaminobenzoic acid) [SEQ ID NO: 36]

To a solution of Fmoc-AAAPV-(diaminobenzoic acid) [SEQ ID NO: 36] (400mg; Example 64) in DMF (3 mL) was added piperidine (0.5 mL). The mixturewas stirred overnight, and concentrated by evaporation (1 mbar, 55° C.).The residue was stirred 2× with diethylether (25 mL) and intermediatefiltration. The product formed was sufficiently pure for subsequent use.ESI-MS (positive) [M+H]=562.

Example 66 Poly(AAAPV-{diaminobenzoic acidjacrylamide) [SEQ ID NO: 36]

Cysteamine hydrochloride (11 mg) was dissolved in DMF (5 mL) pre-treatedwith argon. Poly-4-nitrophenyl acrylate (120 mg) and triethylamine (200μL) were added. Care was taken to exclude oxygen during the entireprocess. The mixture was stirred for 1 h at 55° C.H-AAAPV-(diaminobenzoic acid) [SEQ ID NO: 34] (230 mg; Example 65) wasdissolved with DMF (3 mL), treated with argon and added to the reactionmixture. The mixture was stirred overnight at 65° C. The product wasprecipitated under a stream of argon with methanol (100 mL), containing0.5% formic acid. (Due to the polymeric nature of the materials, nospecific characterization was possible. However, material passed throughSephadex is of high molecular weight and blue, thus proving attachmentof the dye to the polymer.)

Example 67. Poly-co-[(N-mercaptoethylacrylamide)-(AAAPV-{RBB}-acrylamide)] [SEQ ID NO: 36]

[R=Ala-Ala-Ala-Pro-Val-RBB] [SEQ ID NO: 36]

a) Cysteamine hydrochloride (2 mg) was dissolved in DMF (5 mL)pre-treated with argon. Poly-4-nitrophenyl acrylate (36 mg) andtriethylamine (50 μL) were added. Care was taken to exclude oxygenduring the entire process. The mixture was stirred for 1 h at 55° C.H-AAAPV-RBB [SEQ ID NO: 34] (125 mg, Example 68c) was dissolved with DMF(2 mL), treated with argon, and added to the reaction mixture. Themixture was stirred overnight at 65° C. The product was eluted bypassing 1 ml of the reaction mixture with a mixture of water andmethanol (4/1; pre-treated with argon) through a column of Sephadex LH20® (6 g); the blue eluate at the very front was collected andconcentrated in vacuo. (Due to the polymeric nature of the materials, nospecific characterization was possible. However, material passed throughSephadex is of high molecular weight and blue colored, thus provingattachment of the dye to the polymer).

b) 2.2 mL of a solution of 95 mg of poly-p-nitrophenylacrylate in 9.5 mLof DMF was flushed with argon and combined with 46 μL of a solution of32 mg of mercaptoethylamine hydrochloride and 45 μL of trimethylamine in739 μL of argon flushed DMF. After stirring for 30 min, 45 mg ofH-AAAPV-RBB [SEQ ID NO: 34] (example 68c) and 100 μL of triethylaminewere added, and the mixture was agitated for 16h at room temperature.After keeping the mixture at 54° C. for 90 min, 100 μL of ethanolaminewere added and the reaction was kept for further 90 min at 54° C. Theproduct was isolated by passing the mixture through a column of 6 g ofSepahdex LH 20 with water-methanol 3-1. (Due to the polymeric nature ofthe materials, no specific characterization was possible. However,material passed through Sephadex is of high molecular weight and bluecolored, thus proving attachment of the dye to the polymer).

The same procedure can be applied to H-AAAPVV-RBB [SEQ ID NO: 35](example 68c).

Poly-co-[(N-2-{3-(4-azidophenyl)propionyl}acrylamide)-(AAAPV-(RBB)-acrylamide)][SEQ ID NO: 36]

[R=Ala-Ala-Ala-Pro-Val-RBB] [SEQ ID NO: 36]

c) 2.2 mL of a solution of 95 mg of poly-p-nitrophenylacrylate in 9.5 mLof DMF was combined with 2.2 mg of 4-azido phenylalanine and 5.8 μL oftrimethylamine. After agitating for 30 min at RT, 43 mg of H-AAAPV-RBB[SEQ ID NO: 34] was added together with 100 μL of triethylamine, and themixture was agitated for 16h at RT. After keeping the mixture at 54° C.for 90 min, 100 μL of ethanolamine were added and the reaction was keptfor further 90 min at 54° C. The product was isolated by passing themixture through a column of 6 g of Sephadex LH 20 with water-methanol3-1. (Due to the polymeric nature of the materials, no specificcharacterization was possible. However, material passed through Sephadexis of high molecular weight and blue, thus proving attachment of the dyeto the polymer).

The same procedure can be applied to H-AAAPVV-RBB. [SEQ ID NO: 35](Structure as above with R=Ala-Ala-Ala-Pro-Val-Val-RBB).

AAAPV-RBB bound to Beads [SEQ ID NO: 34]

d) 30 mg of H-AAAPV-RBB [SEQ ID NO: 34] was combined with 93 mg ofSephabeads ECEP (epoxy activated) and 50 μL of DMF. The mixture was keptat 54° C. for 72 and washed with dichloromethane-methanol mixture (3-1)and then with DMF, until no more blue eluted.

The same procedure can be applied to H-AAAPVV-RBB [SEQ ID NO: 35]instead of H-AAAPV-RBB [SEQ ID NO: 34]

e) 50 mg of Chemicell SiCore Amin beads were washed with DMF 4 times andsuspended in 800 μL of DMF. 90 mg of poly-p-nitrophenylacrylate wasadded, and the mixture was shaken at 60° C. for 3h. 32 mg of H-AAAPW-RBB[SEQ ID NO: 35] (example 68c) and 50 μL of triethylamine were added.Shaking at 60° C. was continued for 16h. The beads were centrifuged off,the supernatant was decanted and used for example 67 g, and the beadsware treated with 10 μL of ethanolamine in 100 μL of DMF. After washing5 times with 1.5 mL of DMF, the beads were washed 2 times with 1.5 mL ofwater. The beads have acquired a blue color, that can't be washed off.

f) 33 mg of H-AAAPW-RBB [SEQ ID NO: 35] and 19 mg ofpoly-p-nitrophenylacrylate were combined in 0.5 mL of DMF with 50 μL oftriethylamine and agitated at 60° C. for 3h. Beads prepared as in e) (50mg) were added, and shaking at 60° C. was continued for 16h. The beadswere centrifuged off, the supernatant was decanted and used for example67 g, and the beads were treated with 10 μL of ethanolamine in 100 μL ofDMF. The beads were washed 5 times with 1.5 mL of DMF, followed bywashing 2 times with 1.5 mL of water. The beads have acquired a bluecolor, that can't be washed off.

Isolation of Non-Bead-Bound Polymeric Material

g) To the combined supernatants of e) and f) was added 120 μL ofethanolamine. The mixture was kept at 54° C. overnight. After pouring on250 mL of diethylether, containing 1% formic acid, the insolubles wereisolated by centrifugation, dissolved with 2 mL of DMF, and passedthrough a column of 6 g of Sephadex LH 20 with water-methanol (4-1). 2mg of a blue high molecular weight fraction can be isolated.

Example 68. Polymer-Bound Dye with Hydrophobic Anchors and a CleavageSite for Elastase

-   -   R₁=Ala-Ala-Ala-Pro-Val-(RBB [SEQ ID NO: 34] and R₂=H or    -   R₁=n-octyl and R₂=H or    -   R₁=R₂=n-octyl

a) H-AAAPV-RBB [SEQ ID NO: 34] (240 mg; Example 68c) andpoly-(4-nitrophenyl)acrylate (103 mg) were combined and dissolved in DMF(5 mL). Triethylamine (600 μL) was added, and the mixture was agitatedovernight. N,N-Dioctylamine (130 μL) was added and the mixture was keptat 54° C. with occasional shaking for 5 h. n-Octylamine (150 μL) wasadded, and the mixture was kept overnight at 54° C.

The mixture was poured into water (50 mL) containing 1% of formic acid.The precipitated material was collected by centrifugation andre-dissolved with warm methanol (10 mL). After a second precipitationwith water (200 mL, with 0.5% of formic acid), a precipitate appearedand was isolated by filtration. The blue material was dried,re-dissolved with warm methanol (10 mL) and precipitated with diethylether. After filtration and drying, a blue powder remained. (Due to thepolymeric nature of the materials, no specific characterization waspossible).

b) Synthesis of Fmoc-AAAPV-RBB [SEQ ID NO: 36]: A stirred mixture oftetrapeptide Fmoc-AAAP-OH [SEQ ID NO: 32] (740 mg) and HOBt·H2O (200 mg)in 10 ml of DMF was treated with DCC (198 mg) at ambient temperature for15 min. H-V-RBB (680 mg; Example 42) was added in one portion and thereaction mixture was stirred at room temperature until reactionmonitoring via ESI-MS (negative mode) indicated consumption of startingmaterials (˜15 h, overnight). All volatiles were removed under reducedpressure. The solid residue was applied to 10 g of silica gel andchromatographed with a gradient of cyclohexane-ethyl acetate-methanolfrom 7-2-1 to 11-6-5 (each containing 0.1% of formic acid). Yield: 342mg. ESI-MS (negative mode) [M−H]⁻: 1131.

Fmoc-AAAPW-RBB [SEQ ID NO: 37] was prepared using an analogous procedureas Example 68b starting from Fmoc-AAAPW-OH [SEQ ID NO: 38]. ESI-MS(negative mode) [M−H]⁻: 1230.

c) Synthesis of H-AAAPV-RBB [SEQ ID NO: 34]: Fmoc-AAAPV-RBB [SEQ ID NO:36] (342 mg, example 68b) was dissolved with DMF (˜12 mL). Piperidine(0.5 mL) was added. The mixture was stirred for 1 h at room temperature,until reaction monitoring with MS indicated consumption of the startingmaterial (m/z=1131, negative mode) and formation of the product(m/z=909, negative mode). All volatiles were removed under reducedpressure, and the residue was stirred for 30 min with a mixture of DCMand methanol (5:1, 5 ml), and then precipitated by addition of diethylether (20 mL). The precipitate was isolated by filtration and treatedfor 60 min with diethylether (25 mL) with heavy stirring. Afterfiltration by suction and drying at 54° C. for 3 h, 288 mg of an intenseblue, fine powder was isolated. ESI-MS (negative mode) [M−H]⁻: 909.

Synthesis of H-AAAPW-RBB [SEQ ID NO: 35] was prepared using an analogousprocedure as described in Example 68c. ESI-MS (negative mode) [M−H]⁻:1008.

d) 40 mg of AAAPV-RBB [SEQ ID NO: 34] and 14 mg of poly-p-nitrophenylacrylate were combined in 1 mL of DMF. When everything was dissolved,100 μL of trimethylamine are added, and the mixture was agitated for 30min. 20 μL of N,N-dioctylamine are added, and the mixture was agitatedat RT overnight. The reaction mixture was passed through a column of 6 gof sephadex LH 20 with methanol-water mixture (1+3) twice to yield 13 mgof a dark blue polymeric powder. (Due to the polymeric nature of thematerials, no specific characterization was possible. However, materialpassed through Sephadex is of high molecular weight and blue, thusproving attachment of the dye to the polymer).

e) Synthesis of N-[(3-triethoxysilyl)propyl]-N′-[AAAPW-RBB]urea [SEQ IDNO: 37] and subsequent binding of the compound to silica gel:H-AAAPW-RBB [SEQ ID NO: 35] (49 mg; Example 68c) was dissolved with dryDMF (2 mL). (Isocyanatopropyl)triethoxysilane (80 μL as a 10% solutionin dry DMF) was added and the mixture was stirred at ambienttemperature. After 2 h, a second portion of(isocyanatopropyl)triethoxysilane (80 μL as a 10% solution in dry DMF)was added and the mixture was stirred overnight. When reactionmonitoring by ESI-MS indicated complete conversion (negative mode)[M−H]⁻: 1255), 35 mg of silica gel (Reprosil 100, Dr. Maisch, 5 μm, poresize 100 Angström, spec, surface 280 m²/g) was combined with 100 μL ofthe reaction mixture and incubated overnight at 75° C. with shaking. Theproduct was isolated by centrifugation and washed with DMF (5×1 mL) andwater (2×1 mL). It comprised a dark blue powder, that did not leach anyblue with water or DMF within 5 h.

Example 69. Fmoc-A-(N-phenylpiperazinyl)amide

Fmoc-A-OH (1.69 g) and HOBt (1.2 g) were suspended in DCM (25 mL).1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1.12g) was added and the mixture was stirred for 10 min. N-Phenylpiperazine(605 mg) was added and the mixture was stirred for an additional 60 minat room temperature. The mixture was extracted with 1 N aq. HCl and 1 Naq. K₂C0₃ (2× each), then with brine and dried over sodium sulfate.Evaporation yielded 1.3 g of sufficiently pure product. ESI-MS(positive) [M+H]=456.

Example 70.Bis-(4-dimethylaminophenyl)-(4-[4-N-(N-Fmoc-alanyl)]piperazinophenyl)-carbeniumion [Fmoc-A-(CV)] (a) andBis-(4-dimethylaminophenyl)-(4-[N-piperazino]phenyl)-carbenium ion (CV)(b)

To a mixture of crude Fmoc-A-N-phenylpiperazine amide (2.52 g, example69) and 4,4′-bis(N,N-dimethylamino)benzophenone (Michler's ketone, 1.92g), was added phosphorous (V) oxichloride (1.6 mL), and the reagentswere mixed until everything was homogenously wet. The mixture was heatedto 100° C. for 2 h, left to cool and dissolved with a mixture of acetoneand water (1:1). Silica gel (8 g) was applied to the blackish-bluesolution, and the mixture was evaporated to dryness. Elution with agradient of cyclohexane-ethyl acetate-methanol (8/1/1, containing 0.5%formic acid to 3/1/1, containing no acid; then 0/1/1, containing 3%triethylamine) provided 1.22 g of (a) (m/z=706) and 720 mg of (b)(m/z=413) as a salt. ESI-MS (positive) [M]=706 and 413, respectively.

Example 71. 4-([4-(H-Ala-Ala-Pro-Val-Ala) [SEQ ID NO: 28] amidoN-piperazino]phenyl)-bis-(4-dimethyl-aminophenyl)-carbenium ion(H-AAPVA-CV) and 4-([4-(H-Ala-Ala-Pro-Val-Ala)amidoN-piperazino]phenyl)-bis-(4-dimethyl-aminophenyl)-carbenium ionpolyacrylamide

A. Fmoc-A-CV (980 mg; Example 70a) was dissolved with DCM (40 mL).Excess piperidine (2 mL) was added, and the mixture was stirredovernight. All volatiles were removed in vacuo and residual piperidinewas removed in vacuo at <30 mbar/50° C. The residue was washedcyclohexane (3×), dried and dissolved with DCM (10 mL). The product wasidentified by MS (m/z=484).

B. Fmoc-Val-Pfp (870 mg) was added, and the mixture was stirred for 15min. All volatiles were removed in vacuo, and the residue was treatedwith piperidine (2 mL) in DCM (20 mL). All volatiles were removed, andthe mixture was washed with cyclohexane (3×), and dried in vacuo at <30mbar/50° C. The mixture was purified by HPLC to yield 110 mg of thedesired intermediate.

C. Fmoc-AAP-OH (110 mg) was combined with HOBt (50 mg) and suspended inDMF (20 mL). EDCI (50 mg) was added, and the mixture was stirred for anadditional 10 min. The intermediate from step B (110 mg) was added, andthe mixture was stirred for an additional 2 h. Reaction monitoring withMS (m/z=1044) indicated the formation of expected product.

D. The reaction mixture was treated with piperidine (0.5 mL) and stirredovernight. MS indicated formation of the target compound (ESI-MS(positive) [M]=822). All volatiles were removed in vacuo at <30 mbar/50°C. Treatment of the material with elastase released A-CV, as found byMS-analysis (m/z=484, positive mode).

E. 579 mg of poly-p-nitrophenylacrylate are dissolved with 6 ml of DMF.The residue of D is added and the mixture is agitated for 72h at RT. MSindicates disappearance of the peak at m/z=822. 500 μL of ethanolamineare added, and the mixture is agitated at RT for 7h. The blue color ofthe CV-cation can be reestablished by acidic treatment. 1 mL of thepreparation is passed through a column of Sephadex LH 20 (20 mL,water/methanol 2+1). The bluish-green fraction eluting at the front iscollected and dried i.v. Treatment with porcine and human elastasereleases A-CV, as proven by the appearance of a mass peak at m/z=484.(Due to the polymeric nature of the materials, no specificcharacterization was possible. However, material passed through Sephadexis of high molecular weight and blue, thus proving attachment of the dyeto the polymer. Furthermore, the disappearance of the peak at m/z=822 inthe mass spectrum during the reaction supports this).

Example 72.Bis-(4-dimethylaminophenyl)-(4-[4-N-(6-N-valinoyl)aminohexanoyl)]piperazinophenyl)-carbeniumion (Fmoc-Val-Aminohexyl-CV or Fmoc-V-Ahx-CV)

To a solution of C V (720 mg; Example 70b) in DCM (15 mL) was addedFmoc-6-aminohexanoic acid pentafluorophenyl ester (1.18 g). The mixturewas stirred for 1 h prior to the addition of piperidine (1 mL). Afterstirring for 1 d, all volatiles were removed in vacuo, and the residuewas extracted with cyclohexane (3×) and re-dissolved with DCM (15 mL).Fmoc-Val-Pfp (1.13 g) was added, and the mixture was stirred for 1 h.All volatiles were removed, and the residue was dissolved with a mixtureof methanol (22 mL) and water (3 mL) (containing 3% formic acid). Thepreparation was precipitated with water with 3% of formic acid (50 mL)and centrifuged. The residue was separated by preparative FIPLC to yield160 mg of a blackish-blue material. The material was dissolved with DCM(5 mL) and treated with piperidine (250 overnight. Concentration andpurification with FIPLC yielded 40 mg of the desired product. ESI-MS(positive) [M]=847.

Example 73. H-AAPV-Ahx-CV [SEQ ID NO: 21]

Treatment of Fmoc-V-Ahx-C V (Example 71) as described in Example 42,Step D, produced H-AAPV-Ahx-CV [SEQ ID NO: 21]. ESI-MS (positive)[M]=864

Treatment of the product of Example 72 with elastase releases no Ahx-CV,as found by MS-analysis (no m/z=526, positive mode).

Example 74. Jeffamine EDR176-Modified Hypromellose

Hypromellose (89 kDa, 5 g) was dissolved in MP (150 mL). Afterdissolution, toluene (100 mL) was added. The mixture was refluxed with adean-starck trap for 1 h to remove water by azeotropic distillation. Thedry solution was cooled to 0° C. At this temperature, tosyl chloride(1.45 g) was added, followed by pyridine (30 mL). The mixture was keptat this temperature for 1 h. Finally Jeffamine EDR 176 (15 g) was added,and the mixture was stirred for 12h at room temperature. Then themixture was heated to 90° C. for 1 h. After the reaction was complete,the volatiles were removed by evaporation. The resulting solution wasdialyzed against water (MWCO 10.000-20.000 Da). The solution wasevaporated to dryness. The resulting product was dissolved in ethanoland precipitated with ethyl acetate/cyclohexane (3×). The product was adark brown polymer film. Yield: 1.7 g; 0.34 mmol/g N.

Example 75. EDA modified Hypromellose

Hypromellose (89 kDa, 5 g) was dissolved in MP (120 mL). Afterdissolution, toluene (50 mL) was added. The mixture was refluxed with adean-starck trap for 1 h to remove water by azeotropic distillation. Thedry solution was cooled to 0° C. At this temperature, mesyl chloride(1.9 mL) was added, followed by pyridine (20 mL). The mixture was keptat this temperature for 1 h. Finally, ethylene diamine (EDA, 100 mL) wasadded, and the mixture was stirred for 12 h at room temperature. Thenthe mixture was heated to 90° C. for 1 h. After the reaction wascomplete, the volatiles were removed by evaporation. The resultingsolution was dialyzed against water (MWCO 10.000-20.000 Da). Thesolution was evaporated to dryness and the resulting product wasextracted with ethyl acetate (5×). Slightly yellow film. Yield: 2.9 g;0.67 mmol/g N.

Example 76. EDA Modified Hydroxyethyl Cellulose

Hydroxyethyl cellulose (90 kDa, 10 g) was dissolved in NMP (250 mL).After dissolution toluene (200 ml) was added. The mixture was refluxedwith a dean-starck trap for 1 h to remove water by azeotropicdistillation. The dry solution was cooled to 0° C. At this temperaturetosyl chloride (8.8 g) was added, followed by pyridine (3.75 g). Themixture was kept at this temperature for 2 h and at room temperature for5 h. Finally ethylene diamine (30 mL) was added, and the mixture wasstirred for 12 h at room temperature. Then the mixture was heated to 90°C. for 2 h. After the reaction was complete, insoluble material wasremoved by filtration. Then the volatiles were removed by evaporation.The resulting solution was dialyzed against water (MWCO 10.000-20.000Da). The solution was evaporated to dryness and the resulting productwas extracted with ethyl acetate/ethanol (3:1, 3×). Yellow film. Yield:3.85 g; 0.45 mmol/g N.

Example 77. Jeff Amine EDR 176 Modified Hydroxyethyl Cellulose

Hydroxyethyl cellulose (250 kDa, 5 g) was dissolved in MP (160 mL).After dissolution, toluene (100 mL) was added. The mixture was refluxedwith a dean-starck trap for 2 h to remove water by azeotropicdistillation. The dry solution was cooled to 0° C. At this temperature,tosyl chloride (4.2 g) was added, followed by pyridine (1.75 g). Themixture was kept at this temperature for 2 h and at room temperature for2 h. Finally, Jeffamine EDR 176 (20 mL) was added, and the mixture wasstirred for 12 h at room temperature. Then the mixture was heated to 90°C. for 2 h. After the reaction was complete, insoluble material wasremoved by filtration. Then the volatiles were removed by evaporation.The resulting solution was dialyzed against water (MWCO 10.000-20.000Da). The solution was evaporated to dryness and the resulting productwas extracted with ethyl acetate (3×). Yellow film. Yield: 4.1 g, 0.99mmol/g N in the high MW fraction.

Example 78. Trioxatridecandiamine Modified Hydroxyethyl Cellulose

Hydroxyethyl cellulose (90 kDa, 5 g) was dissolved in NMP (200 mL).After dissolution, toluene (150 mL) was added. The mixture was refluxedwith a dean-starck trap for 2 h to remove water by azeotropicdistillation. The dry solution was cooled to 0° C. At this temperature,tosyl chloride (4.2 g) was added, followed by pyridine (1.75 g). Themixture was kept at this temperature for 3 h and at room temperature for4 h. Finally, O,Oalbis(3-aminopropyl)diethylene glycol (50 g) was addedand the mixture was stirred for 12 h at room temperature. Then themixture was heated to 90° C. for 3 h. After the reaction was complete,insoluble material was removed by filtration. Then the volatiles wereremoved by evaporation. After adding water (100 mL), the resultingsolution was dialyzed against water (MWCO 10.000-20.000 Da). Thesolution was evaporated to dryness and the resulting product wasextracted with ethyl acetate (3×). The sticky brown mass was dried overP4010 and again extracted with ethyl acetate/ethanol (3:1). Dark brownfilm. Yield: 5.6 g; 0.98 mmol/g N in the high MW fraction.

Example 79. HEC-EDR176-N-Propionyl-AAPVA-RBB [SEQ ID NO: 26]

Jeffamine EDR 176 modified hydroxyethyl cellulose (0.05 g; Example 77)was dissolved in H₂O (10 mL) and MeOH (10 mL). To this solution,Acrylamido-AAPV A-RBB [SEQ ID NO: 26](Example 49) (21 mg) in MeOH (3 mL)was added. The mixture was adjusted to pH 8.0 with NaHCO₃ and stirred atroom temperature for 18h. A blue precipitate was formed. This mixturewas dialyzed against NaHCO₃ and water (2×), (MWCO 12-14 kDa). Afterfiltration, the light blue solution was evaporated to yield a bluecolored film. Yield: 45 mg, blue color in the high MW fraction.

Example 80. HEC-PEG200-N-propionyl-AAPF-RBB [SEQ ID NO: 12]

Trioxatridecandiamine modified hydroxyethyl cellulose (0.22 g; Example78) was dissolved in H₂O (20 mL) and MeOH (10 mL). To this solution,Acrylamido-AAPF-RBB [SEQ ID NO: 12](example 47 0.085 g) in MeOH (5 mL)was added. The mixture was adjusted to pH 8.0 with NaHCO₃ and stirred atroom temperature for 18 h. A blue precipitate was formed. This mixturewas dialyzed against NaHCO₃ and water (2×). After filtration, the lightblue solution was evaporated to yield a blue colored film. Yield: 137mg, blue color in the high MW fraction.

Example 81. HEC-EDA-N-propionyl-AAPF-RBB [SEQ ID NO: 12]

EDA modified hydroxyethyl cellulose (0.54 g; Example 76) was dissolvedin H₂O (50 mL). To this solution, Acrylamido-AAPF-RBB [SEQ ID NO: 12](example 47) (0.085 g) in ACN (30 mL) was added. The mixture wasadjusted to pH 8.0 with NaHCO₃ and stirred at room temperature for 48 h.The solvent was evaporated under reduced pressure. A blue precipitatewas formed. This mixture was dialyzed against NaHCO₃ and water. Afterfiltration, the light blue solution was evaporated to yield a pale bluefilm that did not release color on washing. Yield: 400 mg.

Example 82. PEI-N-propionyl-AAPF-RBB [SEQ ID NO: 12]

Polyethyleneimine (0.2 g; MW 60-75 kDa) was dissolved in H₂O (10 mL). Tothis solution, Acrylamido-AAPF-RBB [SEQ ID NO: 12] (15 mg) (Example 47)in ACN (1 mL) was added. The mixture was stirred for 18 h. Then thesolution was dialyzed (MWCO 12-14 kDa) against NaHCO₃ and water. Afterfiltration, the solvent was evaporated. A blue sticky residue wasobtained. Yield: 140 mg.

Example 83. Acrylamido-AAPV-RBB [SEQ ID NO: 12]

Synthesis: 420 mg of H-AAPV-RBB [SEQ ID NO: 21] (example 44) and 200 mgof sodium bicarbonate are suspended in 25 mL of DMF. 45 μL of acryloylchloride are added. After 2 h, further 40 μL of acryloyl chloride areadded. After finishing the reaction overnight, the mixture isconcentrated and chromatographed over silica gel with ethyl acetate toremove side products and impurities and then with methanol to elute theproduct. Yield 125 mg, ESI-MS (negative mode) [M−H]⁻: 892.

Example 84. α,ω-Bis(N-Propionyl-AAPV-RBB)PEG_((24k)) [SEQ ID NO: 2]

α,ω-Bis-amino PEG (0.3 g; MW 24 kDa, 0, 106 mmol/g N) was dissolved inH₂O (5 mL). To this solution, Acrylamido-AAPV-RBB (33 mg) [SEQ ID NO: 2](example 83) in ACN (20 mL) was added. The mixture was stirred for 24 hat room temperature. Then the solution was dialyzed (MWCO 12-14 kDa)against NaHCO₃ in water/ethanol (9:1, 2×) and water (2×). Afterfiltration, the solvent was evaporated. A blue water-soluble solidpolymer was obtained wherein dye was associated with the high molecularweight fraction. Yield: 140 mg.

Example 85. α,ω-Bis(N-Propionyl-AAPV-RBB)PEG_((11k)) [SEQ ID NO: 2]

α,ω-Bis-amino PEG (0.25 g; MW 11.4 kDa, 0,190 mmol/g N) was dissolved inH₂O (5 mL). To this solution, Acrylamido-AAPV-RBB [SEQ ID NO: 2] (30 mg)(example 83) in ACN (20 mL) was added. The mixture was stirred for 48hat room temperature. Then the solution was dialyzed (MWCO 12-14 kDa)against NaHCO₃ in water/ethanol (9:1, 2×) and water (2×). Afterfiltration, the solvent was evaporated. A blue water-soluble solid wasobtained in which blue color was associated with the high MW fraction.Yield: 120 mg.

Example 86. Propylamino Pullulan

Cyanoethyl Pullulan (3.5 g) was dissolved in H₂O (200 mL). THF (200 mL)was added. To this solution 6.0 g of CoCl₂*6 H₂O was added. The purplesolution was cooled in an ice bath. At this temperature 6.8 g NaBH₄ wasadded over the course of 6 h. A black precipitate formed. The reactionmixture was stirred for an additional 12 h at room temperature. Themixture was acidified with acetic acid (10 mL). After 1 d the blackprecipitate was dissolved and the resulting purple solution wassubjected to dialysis (MWCO 12-14 kDa, water 3×). A white, viscoussuspension was formed. To isolate the product as free amine, NaOH (˜2 g)was added. The less viscous product was dialyzed against water. Afterevaporation a brown solid formed, which was insoluble in water. Brownsolid, 1.85 mmol/g N. Yield: 2.4 g.

Example 87. Propylamine HEC

HEC (5.0 g; 90 kDa) was dissolved in H₂O (50 mL). NaOH (0.16 g) wasadded. To this solution, acrylonitrile (0.8 mL) was added within 4 h (in0.2 ml portions) and stirred at room temperature for another 12 h. Theyellow solution was dialyzed against water (MWCO 10-20 kDa, 2×). Theresulting solution (150 mL) was diluted with THF (75 mL). CoCl₂·26 H₂O(6.3 g) was added. Reduction was achieved by adding NaBH₄ (6.8 g) within4 h. A black precipitate was formed. The mixture was acidified withacetic acid (5 mL). After dissolution of the precipitate (2 d), thesolution was dialyzed against water (MWCO 10-20 kDa, 2×). Afterevaporation, a yellow water-soluble solid was obtained. Yield: 3.5 g,1.02 mmol/g N.

Example 88. N-(Propionyl-AAPV-RBB)-Propylamino HEC [SEQ ID NO: 2]

Propylamino HEC (500 mg; Example 87) was dissolved in H₂O (50 mL).Acrylamido-AAPV-RBB (75 mg) [SEQ ID NO: 2] (example 83) was dissolved inACN (25 mL). The solutions were combined and stirred for 24 h. Then thereaction mixture was kept at 54° C. for 2 d. The resulting dark bluesolution was dialyzed against NaHCO₃ in water/EtOH (9:1, 2×) and water(2×). After partial evaporation to 15 mL, a highly viscous blue solutionwas obtained. Under vigorous stirring, this solution was added to ACN(80 mL). The resulting blue precipitate was extracted with ACN until nomore unreacted dye was detected in the supernatant. The product wasstored in ACN and was a blue rubber-like solid, soluble in water/MeOH.

Example 89. Propylamine HEC (Scale-Up of Example 87 with LowerConcentration of Cobalt and Catalysts)

Hydroxyethyl cellulose (50 g; 90 kDa) was dissolved in H₂O (500 mL).NaOH (1.6 g) was added. To this solution, acrylonitrile (9.0 mL) wasadded within 6 h (in 2.0 ml portions) and stirred at room temperaturefor another 12 h. The mixture was diluted with water/THF (1:1; 200 mL)and additional acrylonitrile (2 mL) was added. After 1 h stirring atroom temperature, the yellow solution was dialyzed against water (MWCO10-20 kDa, 2×, 36 h). This solution (1160 mL) was diluted with THF (250mL). CoCl₂·6 H₂O (25 g) was added. Reduction was achieved by addingNaBH₄ (44 g) within 18 h at 0-5° C. A black precipitate was formed. Themixture was acidified with acetic acid (50 mL). Within 3 d, the blackprecipitate was dissolved. After dissolution of the precipitate, thesolution was dialyzed against water (MWCO 10-20 kDa, 2×). After partialevaporation of the solvent, a viscous yellow solution was obtained.Solid content: −30 g. Yield: 30 g; 0.85 mmol/g N in dry matter.

Example 90. Acryloylated Long-Chain PEG(2000)-AAPV-RBB Linker. [SEQ IDNO: 2]

100 mg Bis-amino PEG 2000 was dissolved in DMF (10 mL). 12 mgacryloylated AAPV-RBB [SEQ ID NO: 2] (Example 83) in DMF (1 mL) wasadded and the mixture was stirred for 1 h. Then the reaction mixture waskept at 54° C. for 18 h. After evaporating the solvent, diethyl etherwas added to extract excess bis-amino PEG and to precipitate theproduct. The precipitate was washed with ether (2×), dried andre-dissolved in ACN (25 mL). NaHCO₃ (50 mg) and acryloychloride (20 μL)were added and the mixture was stirred for 2 h. The mixture was filteredand evaporated to dryness. After extraction with ether, the residue wasdissolved in water/ACN (1:1, 1 mL). The product, a dark violet solution,was used without further purification.

Example 91. [N-3-(triethyoxsilyl)propylaminocarbonyl]-AAPV-RBB [SEQ IDNO: 2]

211 mg of H-AAPV-RBB (Example 44) was dissolved in 10 mL of DMF. 63 of3-(triethoxysilyl)propyl isocyanate was added in one portion andstirring was continued for 10 min. The solvent was removed and theproduct was precipitated with ether. After washing with ether, a darkviolet powder was obtained. Yield: 120 mg; m/z=1085 (M−H).

Example 92. AAPV-RBB-Labelled Silica [SEQ ID NO: 2]

14 mg of [N-3-(triethyoxsilyl)propylaminocarbonyl]-AAPV-RBB [SEQ ID NO:2] (example 91) was dissolved in 1.2 mL of DMF. 100 mg of Silica gel wasdispersed in this solution. Then 1 μL. of sulfuric acid was added andthe reaction mixture was heated to 75° C. The mixture was kept at thistemperature for 2 days. The dark blue silica gel was transferred into acolumn and washed with acetonitrile (200 mL), water/acetonitrile (1:1;500 mL), water/methanol (1:1; 500 mL), and water (500 mL). Results: 65mg of dark blue powder (see enzyme assay for further characterization).

Example 93. Peptide-Labelled beadBALL Amine Microspheres with Long-ChainLinker

Acryloylated long-chain PEG(2000)-AAPV-RBB [SEQ ID NO: 2] linker (0.5mL; Example 90) was added to a 1-ml beadBALL Amine microspheredispersion. The reaction was performed at 60° C. for 2.5 d. Themicrospheres were then centrifuged and washed with water/ACN (1:1, 8×)and water (2×). The resulting pale violet precipitate was re-suspendedin water (1 mL) to provide a pale violet dispersion.

Example 94. Amidation of Fmoc-AAPV with Dianisidine [SEQ ID NO: 2]

Fmoc-AAPV [SEQ ID NO: 9] (1 16 mg) and HOBt (40 mg) were dissolved inDCM (20 mL). The solution was placed in an ice bath and DCC (45 mg) wasadded. Stirring was continued for 30 min at 0° C. and an additional 30min at room temperature. Then a five-fold excess of o-dianisidine (250mg) was added. After a reaction time of 121h, the product was observedby ESI-MS (positive mode) [M+H]⁺: 805, [M+Na]⁺: 827).

Example 95: Synthesis of O-allyl Chlorophenol Red

Chlorophenol red (2.76 g), allylbromide (1.29 mL), and potassiumcarbonate (1.8 g) were combined in dry acetone (50 mL) and heated for 96h to reflux. After cooling to room temperature, all solids were filteredoff and washed with acetone. The combined filtrates (˜70 mL) were pouredinto diethyl ether (350 mL) with vigorous stirring. Stirring wascontinued for 20 min, then the precipitate was collected by filtrationand re-dissolved (50 mL of acetone) and precipitated (400 mL of diethylether). After stirring for 30 min, the precipitate was collected byfiltration and dried at 54° C. to yield 2.72 g. ESI-MS (negative mode)[M−H]⁻: 461; the material no longer changed color with pH.

The same procedure with bromophenol red affords an analogous product.(ESI-MS (negative mode) [M−H]⁻: 551, isotopic pattern of 2 Br, 64%)

Example 96: Synthesis of C-allyl Chlorophenol Red

O-Allyl chlorophenol red (2 g, Example 95) was suspended in nitrobenzene(10 mL) and heated in an oil bath of 210° C. for 70 min. When TLCcontrol (silica gel, Chloroform-methanol 10-3, 2% of formic acid)indicated complete conversion (educt: Rf=0.65, product: Rf=0.55,starting material does not change color upon exposure to ammonia,product changed color to dark violet upon exposure to ammonia), thereaction was left to cool to RT and then diluted with diethyl ether (50mL). The precipitate was collected by decantation, re-dissolved withmethanol (25 mL) and precipitated by pouring into vigorously stirreddiethyl ether (300 mL). The solution-precipitation procedure wasrepeated until no smell of nitrobenzene was detected (2-3×). The productwas dried at 54° C. to yield the target compound (1.39 g). ESI-MS(negative mode) [M−H]⁻: 461; the material changed color when treatedwith base to deep violet.

Example 97: Synthesis of C-allyl Bromophenol Red

O-Allyl bromophenol red (690 mg, Example 95) was suspended innitrobenzene (10 mL) and heated in an oil bath of 210° C. for 60 min.When TLC control (silica gel, Chloroform-methanol 10-3, 2% of formicacid) indicated complete conversion (educt: Rf=0.59, product: Rf=0.45,educt does not change color upon exposure to ammonia, product changedcolor to dark violet upon exposure to ammonia), the reaction was left tocool to RT and then diluted with diethyl ether (50 mL). The precipitatewas chromatographed over silica gel with chloroform-methanol-cyclohexane(6-1-4) to yield 210 mg of the target compound. ESI-MS (negative mode)[M−H]⁻: 551 (isotopic pattern of 2 Br); the color changed with ammoniato violet.

Example 98: Chemical Entity Containing a pH-Sensitive Moiety

A chemical entity comprising a pH-sensitive moiety selected frombromothymol blue, phenol red, bromophenol red, chlorophenol red, thymolblue, bromocresol green, bromocresol purple; and other sulfophthaleindyes is linked to the anchor region using methods known in the art. Forexample the pH-sensitive moiety is linked to the anchor region (selectedfrom a polystyrene bead, silica gel bead, polysaccharide bead,polyacrylamide bead, cellulose bead, polysaccharide, derivatizedcellulose, polyacrylate, polyethyleneimine, polyacrylamide,UV-activatable group, and peptidoglycan derivative, and a combinationthereof) via a single bond, an alkylene linker, an alkenylene linker,and alkynylene linker, an amide linker, or an amine linker.

Example 99: Testing of Enzyme Hydrolysis of ELA and CATG Substrates

The foregoing example substrates were tested for their rate ofhydrolysis and utility in tests for ELA and CAT activity detection.

Elastase: Assays with pancreatic porcine elastase and human elastasefrom leucocytes were performed in a total volume of 25 μL, containing 1U/mL enzyme, 1 mg/mL to 5 mg/mL substrate (depending on molecular weightand amount of loaded dye) in 100 mM potassium phosphate buffer, pH 7 (oralternatively a 500 mM NaCl, 100 mM sodium acetate pH 7). Appearance ofthe assay mixture was either a clear solution, a suspension or a gel,depending on the properties of the enzyme substrate. Incubation wasperformed at 37° C. After 3 and 20 h, 10 μL samples were taken and theenzyme was denatured by addition of 10 μL MeOH to each sample. Aftermixing and incubation at −20° C. for 20 min the samples were centrifugedat 13.000 rpm for 10 min. Each assay included a control without enzyme,containing 1 mg/mL to 5 mg/mL substrate and analysis was performed inthe same way and in parallel to the enzyme-containing assays. Thesupernatant after MeOH precipitation was analyzed via ion trap MS,either [M+H]+ or [M−H]−, depending on the properties of the molecule andthe expected cleavage products.

Cathepsin: Assays with cathepsin G were performed in a total volume of25 μL containing 0.5 U/mL and, if cleavage was observed—in an additionalassay 0.1 U/mL enzyme, 1 mg/mL to 5 mg/mL substrate (depending onmolecular weight and amount of loaded dye onto the immobile phase) in100 mM Tris-HCl, pH 7 (or alternatively a 500 mM NaCl, 100 mMsodiumacetate mix pH 7). Appearance of the assay mixture was either aclear solution, a suspension or a gel, depending on the properties ofthe enzyme substrate. Incubation, control, sampling and analysis was asabove.

The results of these studies are summarized in Table 2.

TABLE 2 Summary of the rate of cleavage od specific substrates byElastase and Cathepsin Human Porcine Elastase Elatase Cathepsin Ex.Anchor Polymer Spacer Recog-site R-Group Dye Cleavage Cleavage Cleavage 1 Fmoc — — AAPV — Indoxyl +++ n.t. n.t.  2 Fmoc — — AAPV — 5-Bromo-4-+++ n.t. n.t. chloro-indoxyl  3 Fmoc — Ala AAPV — Indoxyl ++ n.t. n.t. 4 Fmoc — Ala AAPV — ++ n.t.  5 Fmoc — Ala AAPV — 1-H Indol-3- ++ n.t.n.t. Amin  6 Fmoc — — AAPV — MeO-napthol + + 0  7 Fmoc — — AAPV —Napthol + + 0  8 Fmoc — — AAPV — 2_Naphthol + ++ 0 12 Fmoc — — AAPF —Indoxyl + + 0 13 Fmoc — — AAA — Indoxyl ++ ++ 0 14 Fmoc — — AAPA —Indoxyl +++ +++ 0 15 Fmoc — — V — Indoxyl 0 0 0 16 Fmoc — — AAAA —Indoxyl 0 0 0 17 Fmoc — — APV — Indoxyl + + 0 18 Fmoc — — Phe — Indoxyl+++ + 0 19 Ac — — Phe — Indoxyl +++ + 0 20 Fmoc — — F-V-T(BzI)-F —Indoxyl 0 0 0 25 Fmoc — — AAPV Ala-Val- RBB n.t. 0 0 1,2,3- triazolyl 26Fmoc — — AAPV 1,2,3- RBB n.t. 0 n.t. triazolyl 27 H — — AAPV Ala-Val-RBB n.t. 0 n.t. 1,2,3- triazolyl 28 H — — AAPV 1,2,3- RBB n.t. 0 n.t.triazolyl 33 H — — AAPV Ala-Ala RBB n.t. ++ 0 37 Fmoc — — AAPF Ala RBBn.t. + n.t. 38 H — — AAPF Ala RBB n.t. +++ 0 39 Fmoc — — AAPV Ala RBBn.t. +++ n.t. 40 H — — AAPV Ala RBB n.t. +++ n.t. 45 Fmoc — — AAPF — RBBn.t. ++ n.t. 46 H — — AAPF — RBB n.t. ++ 0 53 Fmoc — — AAPF — Remazoln.t. + 0 Black-(N′- acetyl)- ethylamid 54 — CMC9M31F — AAPF Ala RBB n.t.++ ++ 55 — CMC9M31F — AAPF — RBB n.t. ++ ++ 56 — paper — AAPV — RBB n.t.0 0 59 Fmoc — Ala AAPM — RBB n.t. 0 0 60 H — Ala AAPM — RBB + 0 0 62aCys(TRT) PAA —C4H8— APPV Ala RBB + + n.t. CH(NH)Boc))— C(═O)—O— CH2—C(═O)— 62b Cys(TRT) PAA —C4H8— AAPV Ala RBB ++ + n.t. CH(NH2)— C(═O)—O—CH2— C(═O)— 63b COOH PAA —C2H4-gly AAPV Ala RBB + ++ n.t. 63c dioctylPAA —C3H6—O— AAPV — RBB + +++ n.t. amine C2—H4—O— C3—H6-gly 63c dioctylPAA —C3H6—O— AAPV Val RBB + +++ n.t. amine C2—H4—O— C3—H6-gly 67amercapto- PAA Ala AAPV — RBB ++ n.t. n.t. ethyl 67a mercapto- PAA AlaAAPV Val RBB +++ n.t. n.t. ethyl 67b mercapto- PAA Ala AAPV — RBB ++n.t. n.t. ethyl 67b mercapto- PAA Ala AAPV Val RBB +++ n.t. n.t. ethyl67d Sepabeads — —CH2— AAPV — RBB 0 n.t. n.t. EP CH(OH)— CH2-Ala 67cSicore- PAA Ala AAPV Val RBB 0 n.t. n.t. amino- Beads 67f Sicore- PAAAla AAPV Val RBB 0 n.t. n.t. amino- Beads 67g hydroxy PAA Ala AAPV ValRBB +++ n.t. n.t. ethyl 68d dioctyl PAA Ala AAPV — RBB ++ n.t. n.t.amine 68d dioctyl PAA Ala AAPV Val RBB +++ n.t. n.t. amine 68e Silica —Si—(CH2)3— AAPV Val RBB +++ n.t. n.t. gel NH—(C═O)- Ala 71e hydroxy PAA— AAPV Ala CV 0(+) n.t. n.t. ethyl 79 — HEC Si—(CH2)3— AAPV Ala RBB +++n.t. n.t. NH—(C═O)- Ala 80 — HEC EDR176-N- AAPF — RBB n.t. ++ +Acrylamido 81 — HEC EDA-N- AAPF — RBB n.t. +++ + Acrylamido 88 — HEC- —AAPV — RBB +++ n.t. n.t. Propyl- amino 92 Silica — Si—(CH2)3— AAPV — RBB++ n.t. n.t. gel NH—(C═O)— 93 Amino- — ECH- AAPV — RBB 0 n.t. n.t. PVC-diamino- beads PEG(2000)

Example 100: Cross-Linked Trap

A quaternary amine polymer such as polyDADMAC was mixed withpolyethyleneimine polymer at a ratio from 19:1 to 5:1 depending on thepreparation. The solvent was a mixture of water and acetonitrile (1:1V/V) and the final concentration of polyamine polymer is 20%. To thiswas added, depending on the preparation, a volume of epichlorohydrincorresponding to any of 3, 6, 9, or 12% VN. The mixture was allowed toreact under reflux at 60 C overnight. Thereafter the mixture is heatedwithout condenser to remove unreacted epichlorohydrin. To this was addedan excess of aqueous ammonia and further reacted to quench all uncreadedepoxides (this latter step may also be left out for reactive bound trapmaterials). The mixture was concentrated in vacuo to remove acetonitrileand epichlorhydrin (if not quenched) and evaporated water was replaced.The final mixture contained 10-40% polyamines W/V. Viscosity wasproportional to the degree of cross-linking.

Example 101: UV Initiated Cationic Super Absorber as Water Sink or DyeTrap

A quaternary amine polymer such as polyDADMAC was mixed with monomerscontaining a quaternary ammonium group such as(3-acrylamidopropyl)trimethylammonium chloride,[2-(Acryloyloxy)ethyl]trimethylammonium chloride, or[2-(Acryloyloxy)ethyl]trimethylammonium chloride in a ration of 5:1,7.5:1, 10:1 or 15:1. To this mixture was added a radical starterselected from benzophenone, phenanthrene quinone, or benzoylperoxide.Additionally a cross linker such as di(trimethylol)propane tetraacrylatewas added at 1% of the molar equivalent of the choline acrylate monomer.The solution was diluted with up to 25% isopropanol. For a dye trap,approximately 1 μL of the solution was applied to an area of paper to anon-woven cloth of 15 mm2 using a rubber stamp. It was then irradiatedunder UV radiation at 254 nM for 20 minutes. Function was demonstratedusing a solution of water containing Brilliant Black 0.5% W/V which wasdrawn along the paper or non-woven and was trapped in the quaternaryamine groups.

As a water sink or as a super absorber, a thicker bed of polymer wasformed. Dots or diameter 4 mm or 4 μL were deposited on a hydrophobicsurface like glass, foam or paper. The deposits were UV hardened using ahand-held UV lamp at 254 nm for 30 minutes. They were then placed into awater suspension of brilliant black dye or bromophenol blue dye. Thedegree of expansion and dye binding indicated potential as a superabsorber or as a dye trap. Superabsorbers are designated via the abilityto absorb greater than 100-fold their dry weight in water.

Using the composition: 1 mL of choline acrylate (80% solution in water),70 μL of di(trimethylolpropane) tetraacrylate, 50 μL of a solution (20%w/v) of benzophenone in 2-propanol, 60 μL of a solution (50% w/v) ofHEPES (4-(2-hydoxyethyl)piperazine-1-ethanesulfonic acid), and 0.3 mL ofpoly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water), deposition on a glass surface andsubsequent irradiation results in flat disks. Applying 4, 6, 10, 20 or30 μL results in disks of approximately similar weight. Water uptake wasas indicated in the following table:

Weight of Weight at 15 Weight at 1440 sample (mg) minutes in water (mg)minutes in water (mg) 4.9 148 1480 6.1 293 1720 9.6 360 2800

Example 102: UV Initiated Cross-Linked Trap and Superabsorber

Synthesis of a Cationic Trap by Polymerization of Acrylates

1 mL of choline acrylate (80% solution in water), 70 μL ofdi(trimethylolpropane) tetraacrylate, 50 μL of a solution (20% w/v) ofbenzophenone in 2-propanol, 60 μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), and 1.77 mL ofpoly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water) were combined and vigorously mixed. 1 μLof this mixture was applied to a solid carrier (glass plate, filterpaper, nonwoven) and irradiated with light of 254 nm for 20 min.

1 mL of (acrylamido)propyl trimethyl ammonium chloride (75% solution inwater), 70 μL of di(trimethylolpropane) tetraacrylate, 50 μL of asolution (20% w/v) of benzophenone in 2-propanol, 60 μL of a solution(50% w/v) of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid),and 1.77 mL of poly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water) were combined and vigorously mixed. 1 μLof this mixture was applied to a solid carrier (glass plate, filterpaper, nonwoven fabric) and irradiated with light of 254 nm for 20 min.

1 mL of choline acrylate (80% solution in water), 70 μL ofdi(trimethylolpropane) tetraacrylate, 50 μL of a solution (20% w/v) ofbenzophenone in 2-propanol, 60 μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), and 3.54 mL ofpoly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water) were combined and vigorously mixed. 1 μLof this mixture was applied to a solid carrier (glass plate, filterpaper, nonwoven fabric) and irradiated with light of 254 nm for 20 min.

1 mL of (acrylamido)propyl trimethylammonium chloride (75% solution inwater), 70 μL of di(trimethylolpropane) tetraacrylate, 50 μL of asolution (20% w/v) of benzophenone in 2-propanol, 60 μL of a solution(50% w/v) of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid),and 3.54 mL of poly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water) were combined and vigorously mixed. 1 μl,of this mixture was applied to a solid carrier (glass plate, filterpaper, nonwoven fabric) and irradiated with light of 254 nm for 20 min.

1 mL of choline acrylate (80% solution in water), 70 μL ofdi(trimethylolpropane) tetraacrylate, 50 μL of a solution (20% w/v) ofbenzophenone in 2-propanol, and 60 μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) were combined andvigorously mixed. 1 μl, of this mixture was applied to a solid carrier(glass plate, filter paper, nonwoven fabric) and irradiated with lightof 254 nm for 20 min.

1 mL of (acrylamido)propyl trimethylammonium chloride (75% solution inwater), 70 μL of di(trimethylolpropane) tetraacrylate, 50 μL of asolution (20% w/v) of benzophenone in 2-propanol, and 60 μL of asolution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) were combined andvigorously mixed. 1 μL of this mixture was applied to a solid carrier(glass plate, filter paper, nonwoven fabric) and irradiated with lightof 254 nm for 20 min.

1 mL of choline acrylate (80% solution in water), 120 μL glycerol1,3-diglycerolate diacrylate, 50 μL of a solution (20% w/v) ofbenzophenone in 2-propanol, 60 μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), and 1.77 mL ofpoly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water) were combined and vigorously mixed. 1 μLof this mixture was applied to a solid carrier (glass plate, filterpaper, nonwoven fabric) and irradiated with light of 254 nm for 20 min.

1 mL of (acrylamido)propyl trimethylammonium chloride (75% solution inwater), 500 μL of trimethylolpropane ethoxylate triacrylate (average Mn˜692), 200 μL of a solution (10% w/v) of 4,4′-dihydroxybenzophenone in2-propanol, and 60 μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) were combined andvigorously mixed. 1 μL of this mixture was applied to a solid carrier(glass plate, filter paper, nonwoven fabric) and irradiated with lightof 254 nm for 20 min.

1 mL of choline acrylate (80% solution in water), 150 μL ofdi(trimethylolpropane) tetraacrylate, 40 μL of a solution (20% w/v) ofbenzophenone in 2-propanol, and 30 mg of triethanolamine were combined,diluted with 350 μL of 2-propanol and vigorously mixed. 1 μL of thismixture was applied to a solid carrier (glass plate, filter paper,nonwoven fabric) and irradiated with light of 254 nm for 20 min.

1 mL of (acrylamido)propyl trimethyl ammonium chloride (75% solution inwater), 300 μL of glycerol 1,3-diglycerolate diacrylate, 200 μL of asolution (10% w/v) of 4,4′-dihydroxybenzophenone in 2-propanol, and 90μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) were combined andvigorously mixed. 1 μL of this mixture was applied to a solid carrier(glass plate, filter paper, nonwoven fabric) and irradiated with lightof 254 nm for 20 min.

1 mL of (acrylamido)propyl trimethyl ammonium chloride (75% solution inwater), 1 mL of trimethylolpropane ethoxylate triacrylate (average Mn˜692), 200 μL of a solution (10% w/v) of 4,4′-dihydroxybenzophenone in2-propanol, 150 μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), and 2 mL ofpoly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water) were combined and vigorously mixed. 1 μLof this mixture was applied to a solid carrier (glass plate, filterpaper, nonwoven fabric) and irradiated with light of 254 nm for 20 min.

As a dye trap, approximately 1 μL of the solutions described above wasapplied to an area of paper or a non-woven cloth of 15 mm2 using arubber stamp. It was then irradiated under UV radiation at 254 nM for 20minutes. Function was demonstrated using a solution of water containingBrilliant Black 0.5% W/V which was drawn along the paper or non-wovenand was trapped in the quaternary amine groups.

Optimal trapping effect was obtained using a degree of deposition thatwas sufficient to trap visible amounts of compound while still allowingwater or protein solutions to pass through the trap area. Using thecomposition I mL of choline acrylate (80% solution in water), 70 μL ofdi(trimethylolpropane) tetraacrylate, 50 μL of a solution (20% w/v) ofbenzophenone in 2-propanol, 60 μL of a solution (50% w/v) of HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), and 0.3 mL ofpoly(diallyldimethylammonium chloride) (molecular weight400,000-500,000, 20% in water) is effective but tends to be overloaded.Dilution to 20 or 40% and deposition of 0.2 to 0.6 μL per 12 mm2 on anon-woven containing viscose followed by 30 minutes irradiation at 254nm with a hand UV lamp resulted in adequate trapping with suitable fluidtransfer. These quaternary amines are most ideally applied as a viscousprinted solution. To this end they can be formulated as follows: 15 g ofa 3.3% Exilva suspension were prepared in demineralized water. 12.5 g ofa 4% PolyDADMAC solution in demineralized water was added stepwise tounder continuous stirring. Then 10.0 g demineralized water was addedstepwise under continuous stirring. Then, 50 g of an aqueous Aerosil 200gel was prepared by hydration of Aerosil 200 in demineralized water (10%WAV). 5 g of a 4% PolyDADMAC solution in demineralized water was addedto the Aerosil gel stepwise. The Aerosil/PolyDADMAC Gel was added insmall aliquots to the Exilva/PolyDADMAC gel and mixed. Finally anadditional 7.5 g of the 4% PolyDADMAC was added to the mixture. Theresulting IOOg solution contained 5% Aerosil200, 0.5% Exilva and 1%PolyDADMAC. This ink formulation can be labelled with 0.002% FluoresceinWAV for revelation under UV254 nm.

Example 103.5-(p-Aminomethyl)phenyl-4-chloro-3-indoxyl-P-D-galactopyranoside

5-Bromo-4-chloro-3-indoxyl-P-D-galactopyranoside (XGal, 100 mg, 0.25mmol) was placed in a round-bottomed flask together with p-aminobenzylboronic acid (38 mg, 0.25 mmol). The species were partially diluted inacetonitrile (3 mL) and water (˜0.5 mL) at ambient temperature whilestirring (magnetic stirrer, 300 rpm). Diisopropyl amine (DIPA, 78 yL,0.55 mmol) was added, followed by palladium(II) acetate (Pd(OAc)₂ 3 mg,0.0125 mmol) and triphenylphosphine species (TPPTS, 21.3 mg, 0.0375mmol) while stirring. The system was heated to 80° C. and treatment wascontinued for 48 h. Reaction progress was monitored by ESI-MS. Aftercooling to room temperature any precipitates were filtered off (desiredproduct stays in solution) and the reaction mixture was directly usedfor further treatment of co-bromo alkylated peptidoglycan derivative.

Example 104:5-(p-Aminomethyl)phenyl-4-chloro-3-indoxyl-P-D-galactopyranoside PGadduct

Dried co-bromo alkylated peptidoglycan (55 mg) was placed in a Eppendorftube and was suspended in an acetonitrile/water (6:1) solution of5-(p-aminomethyl-)phenyl-4-chloro-3-indoxyl-P-D-galactopyranoside (1.5mL, excess) at ambient temperature. The reaction system was shaken for72 h. Afterwards the precipitates were successively washed with waterand ethanol followed by dialytic purification (water, 72 h) to ensureremoval of small molecule XGal species. Upon dialysis the products werefreeze dried to yield a yellow powder.

Example 105: 0-Alkylation of Peptidoglycan with Epibromohydrin

Dried Peptidoglycan (PG) (1.0 g) was placed in a 3 necked round bottomflask and was suspended in 1,4-dioxane (3 ml) at ambient temperaturewhile slowly stirring (magnetic stirrer). Epibromohydrin (300 μL, 3.5mmol) was added, followed by addition of perchloric acid 70% (30 μL).Treatment at ambient conditions was continued for 3 h. Afterwards thereaction mixture was poured into a paper filter and was subsequentlywashed with water, 1,4-dioxane and diethyl ether. Upon filtrationalkylated PG was dried in vacuo and kept in refrigerator for furthertreatment. Yield: >1,06 g. (overall yield cannot precisely bedetermined).

Uses for the preparation include the immobilization of chromogenicsubstrates such as that in example 104 and FIG. 7A-C. Alternatively, theactivated PG may be used to bind a secondary enzyme such as aglucosidase, galactosidase, mannosidase, esterase or phosphatase.

The general method for forming such materials is to take the freshlyactive product of PG and epibromhydrin and then wash it with severalchanges of water and buffer. Then it is mixed with a limiting amount ofglucosidase, galactosidase, mannosidase, peroxidase, esterase orphosphatase and allowed to react under agitation. After reaction it iswashed in an ammonium acetate buffer to quench the remaining Br groupsand remove non-immobilised enzyme.

To perform the assay for lysozyme, PG containing a covalently boundsubstrate is mixed with a PG bound to the cognate accessory enzyme. Ondegradation by lysozyme, the accessory enzyme becomes available toactivate the bound substrate which is also made more soluble by theaction of lysozyme. Thus, PG-X-Gal conjugate is mixed with a PG bound toB-galactosidase.

Example 106: Formation of Chlorinated β-lactam Precursor

3-Chloromethyl-7-(2-phenylacetamido)-3-cephem-4-carboxylate (1.04 g,2.14 mmol) was suspended in DCM (dry, 40 mL) and m-chlorperbenzoic acid(77%, 1.09 g, 4.88 mmol) was added in one portion at RT. The mixture wasstirred at RT over the weekend. The solid was filtered off and washedwith DCM (30 mL); the filtrate was concentrated to dryness. The crudeproduct was taken up in Et₂O (40 mL) and stirred in an ice-bath for 2 h.Then the solid was filtered off and washed with Et₂O (50 mL). Theproduct (811 mg, 73%) was obtained as a white solid.

ESI-MS (positive): [M+Na]⁺=541

Example 107: Formation of β-lactam indoxyl ether

3-Chloromethyl-7-(2-phenylacetamido)-3-cephem-4-carboxylate (488 mg,2.00 mmol) was suspended in acetone (dry, 5 mL) at RT and the mixturewas stirred at RT while streaming argon through it for 10 min. Then5-bromo-4-chloro indoxyl (231 mg, 1 mmol) was added in one portion atRT. Stirring with argon stream was continued for 5 min before potassiumcarbonate (278 mg, 2 mmol) was added at RT in one portion. The mixturewas stirred at RT while passing argon through it for another 10 min.Then additional acetone (dry, 2 mL) was added. After two more minutesstirring at RT with argon stream the mixture was stirred under argonatmosphere at RT overnight. DCM (50 mL) and water (40 mL) were added;after extraction the whole mixture was filtered via a fluted filter.After filtration organic and aqueous phase were separated, the organicphase was dried (Na₂S04) and concentrated to dryness. The crude productwas purified by column chromatography (silica gel, eluent: 2% MeOH inDCM). The fractions containing the product were collected to yield 228mg of a deep brown solid.

ESI-MS (positive): [M+Na]⁺=718

Example 108: Methoxy Aniline Derivative Salts

The substance of example 10 can be a direct substrate of MPO, however,it provides the most rapid reaction when it is formulated as an ion pairof the alkylated reaction product and a methoxy aniline and a divalentanion (see FIG. 12 ). The ideal ratio by mass is circa 2:8 aniline:product of example 10. The most efficient reaction occurs when thedivalent anion is sulphuric acid, however, disulfonic substitutedaromatic systems and phosphoric acid can also be used with diminishedrate. HC1 provides only limited reaction. On reaction, one of theproducts of MPO oxidation appears to be the dimer of the two anilinecomponents (see FIG. 13 ).

A major requirement of such substrates is that they are not reactivewith heme. Various methoxy anilines can be used as alternatives to FastBlue, however, they (3,4-Dimethoxyaniline 2,5-Dimethoxyaniline) tend toreact to a chromophore alone with heme and peroxide. To avoid this,deactivation via the amide is required. A variety of substituents arefeasible. The alkyl anchoring moiety can be replaced by other similarlylipophilic groups either by direct alkylation or variants of theepoxidation reaction of Example 10. Similarly, the amide group can bevaried

Insert Markush Diagram—Fast Blue

Example 109: Preparation of Substrates on Solid Material Like Paper orNon-Woven Materials

Elastase detection: Filter paper circles (6 mm) were impregnated with aimpregnation dispersion mixture (0.25% (w/w) Nonidet, 2% (w/w) decanolin 0.05 M borate buffer pH 8 for 1-2 min. Thereafter the filter paperswere placed on a glass plate and dried for 1-2 h at 54° C. After drying,elastase-substrate FmocAAPV Indoxyl ester from example 1 (20 mg/mL inmethanol) was pipetted on the circles 2 times in 2 μl. steps until afinal amount of 80 μg per test circle (20 mm2) was applied.

Alternatively elastase substrate FmocAAPV Indoxyl ester [SEQ ID NO: 2](example 1) can be mixed in methanol with4-Diazo-3-methoxydiphenylaminsulfate and/or2-Methoxy-4-morpholinobenzendiazonium salt with final concentrations of10 mg/mL FmocAAPV Indoxyl ester, [SEQ ID NO: 2] and 5 to 10 mg/mL of therespective Diazonium salt (or a combination of both). The mixture waspipetted on the impregnated test circles 2 times in 2 μL steps.

MPO detection: Filter paper circles (diameter 6 mm) were impregnated bypipetting of 2 μL of a 40 mg/mL solution of the material of example 108in DMSO Methanol (1 part DMSO, 2 parts MeOH) followed by a drying step(48 h, room temperature). To this are added 10 μg glucose and 3 μL of0.1% glucose oxidase (3 μg) in water.

The positive control indicating moisture contact is a pH indicator basedon a preparation of bromothymol blue in chitosan, containingglutaraldehyde. The mixture is pipetted in the reporter area, afterdrying leading to a dark yellow indicator system.

Alternatively a 0.1% bromocresol purple solution in Ethanol can be used,therefor 1.4 μL is pipetted on a filter paper disc (diameter 6 mm) anddried at 50°-60° C. for 1.5 h.

These various filter papers can be placed onto a stick to allowsimultaneous assessment of bodily fluids at the point of care. We willrefer to these as dipsticks.

Example 110: Clinical Assessment of Airway Aspirates

The dipsticks of Example 109 are maintained in an active state throughappropriate storage and delivered to a clinical facility for testing.They may be used to assess the state of infection in airway aspirates.To test their ability to detect infection in these materials, the sticksare used to assess the aspirates of patients who are intubated as partof intensive care procedures. Aspirates are taken as part of routinecare. After aspiration, the resulting material is placed in contact withthe dipstick and allowed to react for up to 6 minutes. Depending on thedegree of reaction, various cut-off values can be obtained that indicatethe putative concentration of the biomarker enzymes. The presence ofElastase or MPO in the aspirate above background levels is an indicatorof potential infection. To interpret the utility of these data,responses to the dipsticks were compared with what was known clinicallyabout the samples. In some cases the samples were known to be taken froma patient being treated for an infection. In other cases the patient wasconsidered not to have an airway infection. In certain cases, thesamples were taken from a patient who subsequently developed aninfection within two days. These samples were considered “non-infected”because that was the clinical diagnosis at the time they were taken.However, in future studies, we will classify samples from patients whogo on to develop infections within two days as being “infected”.

A total of 52 patients were assessed via 117 samples. The data can betabulated according to the degree of reaction to each biomarker and thecut-off value used to indicate infection. These data were summarized inthe following data sets.

Analysis of elaste response in all 117 patient samples.

Clinical diagnosis pneumonia Sens Spec PPV NPV Elastase as a Yes NoTotal (%) (%) (%) (%) AUC Minute 6 HNE Cut-off 1, 2 Positive 16 19 35100.0 81.2 45.7 100.0 0.906 Negative 0 82 82 Cut-off 2, 3 Positive 16 319 100.0 97.0 84.2 100.0 0.985 Negative 0 98 98 Total 16 101 117Abbreviations: HNE = Human Neutrophil Elastase; MPO = MyeloPerOxidase;Sens. = Sensitivity; Spec. = Specificity; PPV = Positive PredictiveValue; NPV = Negative Predictive Value; AUC = Area Under the Curve.

Analysis of 52 patients using both biomarkers

Clinical diagnosis pneumonia Sens Spec PPV NPV Yes No Total (%) (%) (%)(%) AUC HNE_6_2, 3 + Positive 9 4 13 100.0 90.7 69.2 100.0 0.953MPO_6_2, 3 Negative 0 39 39 HNE_6_2, 3 + Positive 9 3 12 100.0 93.0 75.0100.0 0.965 MPO_6_2, 3 Negative 0 40 40 Total 9 43 52 Abbreviations: HNE= Human Neutrophil Elastase; MPO = MyeloPerOxidase; Sens. = Sensitivity;Spec. = Specificity; PPV = Positive Predictive Value; NPV = NegativePredictive Value; AUC = Area Under the Curve.

These data are an analysis of the biomarker response in which thebiomarker is reported with the time of reaction and the cut-off used.Thus, HNE 6 2,3 means that the reaction was watched for 6 minutes anddegree of reaction in the 4^(th) spot of the example in FIG. 10B_is usedto indicate the presence of a positive sample. Using a higher cut-offfor MPO (3,4) supports the finding.

However, a number of patients were sampled as an infection wasdeveloping and were classified as “uninfected”. An example of thisphenomenon is illustrated in FIG. 14 . In the graph depicted in FIG. 14, the day on which a clinical infection was diagnosed is indicated asTO. T-I and T-2 are the days of sampling prior to the infection beingdiagnosed clinically.

The data shown in FIG. 14 suggest that the response of the biomarkerscan indicate the impending infection before it is clinically apparent toICU staff. These data also suggest that the chromogenic substrates areable to usefully indicate abnormal levels of enzyme markers in an airwayaspirate that is consistent with their infection status.

Example 111: Clinical Assessment of Negative Pressure Wound TherapyFluids

Wound healing may be mediated by applying a vacume to the wound area.This may have many effects including drawing serous fluid from the bodythrough the wound and thus bringing with it growth factors, immune cellsand nutrients. It also may encourage or stimulate granulating tissue toproliferate. The fluids that emerge from the process are an indicator ofthe status of the wound bed. Assessing the presence of infectionbiomarkers may provide an indication if the wound is in good conditionwithout removing the dressing or vacume sponge.

To assess the validity of this approach we took samples from such vacumeacquired fluids and tested the response of the dipstick. The result isconsistent with what is known about the wound and the observations madein example 110. Namely that there is a clear biomarker response in casesof diagnosed or suspected infection (See FIG. 10 A).

Example 112: Placement of the Indicator Materials in Tubing Used inMedical Devices

In the previous two examples, the fluids were taken from the source andanalysed using a dipstick system to estimate biomarker response.However, it is also possible to place materials containing the markersin situ in a medical device. These materials can be considered a form ofmonitoring system. For this application, the materials need to besuitable for sterilizing and they need to respond over the appropriatetime-scale. For example, for many uses such as vacume treatment ofwounds or ventilators, a period of 24h would be sufficient to indicate apotential change in status. To this end, the rapid reaction of thedipstick is not required.

The format is also different. In FIG. 9 , various formats are indicated.In FIG. 9B a format is shown that can accommodate the filter papercircles described in Example 109. The format may, however, be simpler inthat tubing and cylindrical holders may be converted to hold paper ornon-woven materials, polyolefins or celluloses of other inert materialsthat are carriers or solid supports for the reactive substrates (seeFIGS. 9A, C and D).

To test this concept, we prepared hold paper or non-woven materials,polyolefins or celluloses treated with the materials described inExample 109 with some modification. In particular, the Elastasesubstrate was not printed or applied with the diazonium salt. This isbecause rapid reaction is not required in this setting. The conversionof indoxyl to a chromophore is usually via oxidation and the diazoniumsalts provide a direct reaction without the need for oxygen. In themonitoring setting, the reaction time is not critical. Similarly, forMPO oxygen is important and where this is limiting, the reaction rate islimited.

In this format, the passage of fluid gives rise to a wetting of thesupport material and conversion of the substrate. The convertedsubstrate is then visible as a stripe. The presence of the stripe withina certain period of time serves as an indicator of the fact that thesource of the fluid may have become infected. Such a color indicatorcould be place in a vacume line where it comes into contact with a bodyfluid. It may also be place in an aspiration line, or in a ventilatordevice.

Example 113: Electronic Monitoring of the Biomarkers

The color generating substrates are a means to allow an interpretationof biomarker response without use of a device. However, in certaincircumstances, on-line monitoring, or quantitation may call for anelectronic assessment. There are a range of modes that may be employedto this end.

Color sensing is one means in which a sensor can be placed to read thecolor of reflected light and thus the rate of change of the reagents.Color measurement using sensors, however, relies on the fluids havingonly limited background color or high contrast relative to the samplecolor. To achieve color sensing an electronic board is placed oppositethe reagents on the dipstick or chip system. The board contains a sourceof light (e.g. small LEDs with red green and blue color) and a sensingchip. Signal change over time is recorded to estimate the activity inthe sample.

Another mode is amperometric sensing. This is relevant to substratesthat produce or consume a REDOX active product like indoxyl (lysozyme,elastase) or a peroxide (MPO). Oxidation of the substrate gives rise toa current that can be measured by appropriate electrodes. Examples ofREDOX active agents released by the enzyme include indoxyl referred tomany times herein, but also ferrocene as in FIG. 11 .

Example 114: Solubility of Indicator Substrates

The ability of enzyme substrates to stay attached to solid phases suchas paper is important to their function. To test the degree to which thedried substrate resists the re-solution into an aqueous phase, thesubstrates are applied to the solid phase using an appropriate solvent(Acetone, methanol, DMSO etc. see also Example 109) and allowed to dryat room temperature for up to 48 hours. Thereafter, the paper ornon-woven material is incubated in 50 mL of either water or artificialwound fluid (2% bovine serum albumin in phosphate buffered salinecontaining potassium chloride, urea pH 7.2) for 2 minutes with gentleagitation by rotation, thereafter it is washed again using successivechanges of fluid, for example, 4 changes of 50 mL. The solution or lossof the substrate is estimated by a number of methods. These includeLCMSMS analysis of the solving solution or remnant analysis of thematerial on the solid phase by enzymatic reaction or direct analysis byLCUV or LCMSMS. The LCMS methods are as follows:

HPLC grade methanol, acetonitrile, water DMSO and THF were used toprepare stocks and as mobile phase. The quantifications were carried outon an Agilent 1260 series HPLC system with ABSciex API 4000 massspectrometer as a detector. The column used was a ReproSil Pur Phenyl 3μm 60×2 mm, operated at 45° C. The mobile phase was: water+0.1% formicacid and acetonitrile. The chromatographic run was a gradient the flowrate 500 μL/min. The injection volume was 5 μL The parent/fragmentspecies detected were 694/561 for product of example 1, 485/467 for theproduct of example 10 and 273/258 for Fast Blue.

% Solvent A % Solvent B Minutes (Water + 0.1% FA) (acetonitrile) 0 99 11 99 1 2 0 100 5 0 100 6 99 1 10 99 1

10 mg/ml stock standard solutions were prepared for each of thecompounds in methanol, 1:3 mixture of DMSO Methanol and a 1:1 mixture ofDMSO Methanol for each analyte respectively. The stock was dissolved1:100 in THF and further 1:10 dilutions were prepared in THF for acalibration range of 0.1-10000 ng/mL.

Assay by enzymatic reaction is according to Example 109 in which theother required reagents are added to the material after the substratehas been rinsed and dried. Once the required accessory reagents (buffer,activators) are added, an excess of enzyme is added. Reaction is thencompared with unwashed samples. Ideal substrates or substratepreparations resist 2 or more changes of 50 mL of water withoutsubstantial loss of reactivity.

This experimental system defines the “water resistance” of the substratewhen dried on filter paper. Thus, a water resistance of 1 means that asignal is still visible after 1 change of 50 mL of artificial woundfluid. A water resistance of 2 means that the signal is still visibleafter 2 changes of 50 mL of artificial wound fluid and so on.

Example 115: Protease Substrates

Substrates for Cathepsin may be prepared using the methods of earlierexamples for peptide indoxyl coupling. Sequence variation is used tovary enzyme selectivity.

Example 116: Protease Substrate Preparation

Peptide substrates such as those described here can be most efficientlyprepared by coupling di-peptides. Preparation of the dipeptides AA andPV in protected form allows their subsequent direct coupling to form thedesired AAPV product [SEQ ID NO: 5].

Example 117: Viral Protease Substrate Preparation

In certain instances, pathogens are not bacteria but virus. In the upperairway, the majority of infections are viral in nature. In certaininstances, a viral infection resolves without invoking bacterial superinfection. However, it is common that bacteria are also promoted in thecontext of a viral infection. This is because virus often suppress theimmune system during infection and then promote inflammatory responsesin order to better distribute (mucous, coughing and sneezing).

Virus either use self coded proteases, or host proteases. Virus typesthat encode their own proteases employ analogs of the 3 C protease.

The consensus recognition motif for 3C protease isLeu-Glu-Val-Leu-Phe-Gln [SEQ ID NO: 40] motif or LEVLFQ [SEQ ID NO: 40].Thus, one means to assess a sample of mucous for the presence of viralsignals is to use a chromogenic substrate for a viral protease. One suchexample is the peptide LEVLFQ-Indoxyl in which the C-terminal isesterified to indoxyl.

In contrast, many virus types do not encode their own proteolyticfunctions but instead use the host functions. Common is the use of thefurin protease by virus. The consensus furin cleavage site (-RRRR- [SEQID NO: 41] or -RRKR [SEQ ID NO: 42] or RLGR [SEQ ID NO: 43] or LLGR [SEQID NO: 44] or LLAR [SEQ ID NO: 45]) is highly basic. Chromogenic Furinsubstrates cannot be conveniently formed from poly arginine, andinstead, lysine replacement is used. One example of a chromogenicsubstrate is LLAR-indoxyl [SEQ ID NO: 45] or LLAL-indoxyl [SEQ ID NO:45]. Alternatively, arginine is left protected by the Tosyl groups whichare the sterically least cumbersome of the protecting groups availablefor arginine.

While preferred embodiments of the disclosed technology have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosed technology. It should beunderstood that various alternatives to the embodiments of the disclosedtechnology described herein may be employed in practicing the disclosedtechnology. It is intended that the following claims define the scope ofthe disclosed technology and that methods and structures within thescope of these claims and their equivalents be covered thereby.

1-80. (canceled)
 81. A chemical entity comprising a compound of FormulaI:A-R—I   Formula I wherein A is an anchor; R is an enzyme recognitionsite; and I is an indicator region; wherein the enzyme recognition site(R) comprises at least one enzyme-labile region or enzyme-reactiveregion; and wherein the anchor (A) enables binding of the chemicalentity to a support material.
 82. The chemical entity of claim 81,wherein the anchor (A) is covalently or non-covalently associated withthe indicator region (I).
 83. The chemical entity of claim 81, whereinthe enzyme recognition site (R) is specific for a wound-specifichydrolase.
 84. The chemical entity of claim 81, wherein anchor (A)comprises a polysaccharide, a cellulose, a polyacrylate, apolyethyleneimine, a polyacrylamide, a peptidoglycan, or a chitosan, ora monomer thereof, an oligomer thereof, a derivative thereof, a mixtureor a combination thereof.
 85. The chemical entity of claim 81, whereinthe enzyme recognition site (R) comprises a site for protease or a siteelastase.
 86. The chemical entity of claim 85, wherein the enzymerecognition site (R) comprises the amino acid sequence (a)X_(y)AAPX_(y)-Z; (b) X_(y)AAPX_(y)-L-Z; (c) X_(y)AAP(V/F/A)X_(y)-Z or(d) X_(y)AAP(V/F/A)X_(y)-L-Z; wherein each X is, independently, anyamino acid; y is, each, independently a number selected from 0 to 200; Lis a linking moiety; and Z is a label, optionally wherein y is, each,independently an integer from 1 to 50, optionally from 1 to 10,optionally from 1 to 6, optionally wherein each amino acid sequence(a)-(d) is labile for elastase.
 87. The chemical entity of claim 84,wherein the enzyme recognition site (R) comprises a site for 3Cprotease.
 88. The chemical entity of claim 87, wherein the 3C proteaseenzyme recognition site (R) comprises an amino acid sequenceX_(y)UUUU_(y)-Z, wherein X is, each, independently any amino acid; y is,each, independently, a number selected from 1 to 50; U is an amino acidselected from LEVLFQ, and Z is a label, optionally wherein the aminoacid sequence X_(y)UUUU_(y)-Z is labile for 3C protease.
 89. Thechemical entity of claim 84, wherein the enzyme recognition site (R)comprises a site for protease which is cathepsin G, optionally whereinthe cathepsin G protease enzyme recognition site (R) comprises an aminoacid sequence (a) X_(y)N⁴N³N²N¹X_(y)-Z; or (b) X_(y)N⁴N³N²N¹X_(y)-L-Z;wherein each X is independently any amino acid; each y is independentlya number selected from 0 to 6; N⁴ is selected from alanine, glycine,valine, and glutamine; N³ is selected from alanine, glycine, proline,lysine, and serine; N² is selected from proline, alanine, and glycine;N¹ is selected from serine, lysine, phenylalanine, arginine, leucine,and methionine; L is a linking moiety, and Z is a label.
 90. Thechemical entity of claim 81, wherein the indicator region (I) comprisesa dye containing a sulfonylethyl-hydrogensulphate-reactive-group or adye containing a dichlortriazine reactive-group.
 91. The chemical entityof claim 90, wherein the dye containing asulfonylethyl-hydrogensulphate-reactive-group is reactive black 5,remazol brilliant blue, reactive violet 5 or reactive orange 16 or acombination thereof.
 92. The chemical entity of claim 90, wherein thedye containing a dichlortriazine reactive-group is reactive blue 4,reactive red 120, reactive blue 2, reactive green 19, or reactive brown10, or a combination thereof.
 93. The chemical entity of claim 81,wherein the indicator reign (I) comprises a detectable label selectedfrom the group consisting of a luminescent molecule, a chemiluminescentmolecule, a fluorochrome, a fluorescent quenching agent, a lipid, acolored molecule, a radioisotope, a scintillant, biotin, avidin,streptavidin, protein A, protein G, an antibody or a fragment thereof, apolyhistidine, Ni2+, a Flag tag, a myc tag, a heavy metal, and anenzyme.
 94. The chemical entity of claim 81, wherein the enzymerecognition site (R) is provided via a bond between the anchor and theindicator.
 95. The chemical entity of claim 94, wherein the enzymerecognition site (R) is specific for a lactamase or a glycosidase. 96.The chemical entity of claim 94, wherein the enzyme recognition site (R)comprises a lactam linkage comprising a conjugated lactone and amide.97. The chemical entity of claim 94, wherein the glycosidase isβ-lactamase.
 98. The chemical entity of claim 81, wherein the enzymerecognition site (R) comprises a peptide of from four to eight aminoacids, the peptide having an N-terminus and a carboxy terminus; whereinthe anchor (A) comprises a hydrophobic entity having at least four CH₂groups linked to the N-terminus of the peptide; and wherein theindicator region (I) comprises an alcohol-containing or amine-containingchromophore or chromophore precursor linked via an amide or ester orcarbonate bond to the carboxy terminus of the peptide.
 99. The chemicalentity of claim 81, wherein the indicator region (I) comprises adetectable label selected from the group consisting of indoxyl and FastBlue.